miR-17-3p Down-regulates Mitochondrial Antioxidant Enzymes and Enhances Radiosensitivity of Prostate Cancer Cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-18 Zhi Xu, Yanyan Zhang, Jiaji Ding, Weizi Hu, Chunli Tan, Mei Wang, Jinhai Tang, Yong Xu
Radioresistance remains to be a major obstacle in the management of patients with advanced prostate cancer (PCa). We have identified a mature miR-17-3p processed from the 3’-arm of precursor miR-17, which appeared to be able to inhibit three major antioxidant enzymes located in mitochondria, i.e. manganese superoxide dismutase (MnSOD), glutathione peroxidase 2 (Gpx2) and thioredoxin reductase 2 (TrxR2). Here, we show that up-regulation of miR-17-3p remarkably sensitized PCa cells to ionizing radiation (IR). Reductions of the three antioxidants led to increasing cellular ROS accumulation as well as declining mitochondrial respiration. The miR-17-3p mediated dysfunction of mitochondrial antioxidants apparently sensitizing IR therapy was manifested in vitro and in vivo. Substantially, the miR-17-3p effect on suppression of the antioxidants can be efficiently eliminated or attenuated by transfecting with either a miR-17-3p inhibitor or each of the related antioxidant cDNA expression construct. Overall, in addition to the insights into the functional assessments for the duplex of miR-17-5p and miR-17-3p, the present study highlights the rigorous evidence that demonstrated suppression of multiple mitochondrial antioxidants by a single miRNA, thereby providing a promising approach to improve radiotherapy for advanced PCa by targeting mitochondrial function.
Comprehensive RNA sequencing analysis in serum and muscle reveals novel small RNA signatures with biomarker potential for Duchenne Muscular Dystrophy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-17 Anna M.L. Coenen-Stass, Helena Sork, Sole Gatto, Caroline Godfrey, Amarjit Bhomra, Kaarel Krjutškov, Jonathan R. Hart, Jakub O. Westholm, Liz O’Donovan, Andreas Roos, Hanns Lochmüller, Pier Lorenzo Puri, Samir EL. Andaloussi, Matthew J.A. Wood, Thomas C. Roberts
Extracellular small RNAs (sRNAs), including microRNAs (miRNAs) are promising biomarkers for diseases such as Duchenne Muscular Dystrophy (DMD) although their biological relevance is largely unknown. To investigate the relationship between intra- and extracellular sRNA levels on a global scale we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx mice, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, snRNA and piRNA loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance towards wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers.
The IS2 element improves transcription efficiency of integration-deficient lentiviral vectors (IDLVs) episomes. Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-18 Sabina Sánchez-Hernández, Alejandra Gutierrez-Guerrero, Rocío Martín-Guerra, Marina Cortijo-Gutierrez, María Tristán‐Manzano, Sandra Rodriguez-Perales, Laura Sanchez, Jose Luis Garcia-Perez, Jesus Chato-Astrain, Ricardo Fernandez-Valades, Ana Belén Carrillo-Galvez, Per Anderson, Rosa Montes, Pedro J. Real, Francisco Martin, Karim Benabdellah
Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episomes levels in IDLVs-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLVs LTR circles 6-7 fold. The final effect the IS2 element in IDLVs will greatly depends on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1LTR versus 2LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions.
Phenotypic miRNA screen identifies miRNA-26b to promote the growth and survival of endothelial cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-18 Andrea Martello, David Mellis, Marco Meloni, Alison Howarth, Daniel Ebner, Andrea Caporali, Ayman Al HajZen
Endothelial cell proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here, we conducted high content screen of a human miRNA mimic library to identify novel regulators of endothelial cell growth systematically. Several miRNAs were nominated that enhanced or inhibited endothelial cell growth. Out of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human endothelial cell types. miR-26b overexpression enhances endothelial cell proliferation, migration, and tube formation while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of endothelial cells. A combinatory functional siRNA screening of 48 predicted gene targets revealed that miR-26b enhanced endothelial cell growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances endothelial cell proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease.
Tissue-dependent expression and translation of circular RNAs with recombinant AAV vectors in vivo Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-18 Rita M. Meganck, Erin K. Borchardt, Ruth M. Castellanos Rivera, Miranda L. Scalabrino, Jeremy E. Wilusz, William F. Marzluff, Aravind Asokan
Circular RNAs (circRNAs) are long-lived, covalently closed RNAs that are abundantly expressed and evolutionarily conserved across eukaryotes. Possible functions ranging from miRNA and RNA binding protein sponges to regulators of transcription and translation have been proposed. Here we describe the design and characterization of recombinant adeno-associated viral (AAV) vectors packaging transgene cassettes containing intronic sequences that promote backsplicing to generate circularized RNA transcripts. Using a split GFP transgene, we demonstrate the capacity of vectors containing different flanking intronic sequences to efficiently drive persistent circRNA formation in vitro. Further, translation from circRNA is efficiently driven by an internal ribosomal entry site (IRES). Upon injecting AAV vectors encoding circRNA in mice, we observed robust transgene expression in the heart, but low transduction in the liver for the intronic elements tested. Expression in the murine brain was restricted to astrocytes following systemic or intracranial administration, while intravitreal injection in the eye yielded robust transgene expression across multiple retinal cell layers. These results highlight the potential for exploiting AAV-based circRNA expression to study circRNA function and tissue-specific regulation in animal models, as well as development of therapeutic platforms using this approach.
Suppression of IGF1R in Melanoma Cells by an Adenovirus-Mediated One-Step Knockdown System Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-15 Haoran Xin, Mingxing Lei, Zhihui Zhang, Jie Li, Hao Zhang, Xinwei Luo, Aoyun Wang, Fang Deng
Abnormal activation of IGF1R signaling pathway accelerates melanoma development and metastases. RNA interference systems with complex cloning procedures and unsatisfactory efficiency in suppressing gene expression, have become the technical difficulties that hinder their utility to do gene knockdown study. Here we established a simplified adenovirus-mediated gene knockdown system, by which a single adenoviral vector carries multiple siRNA fragments that can effectively suppress IGF1R expression in melanoma cells. We first generated the adenovirus that simultaneously expresses three human or mouse siRNAs targeting IGF1R (AdRIGF1R-OK). Quantitative real-time PCR and immunofluorescence staining revealed that IGF1R expression was significantly decreased in the melanoma cells that were infected with AdRIGF1R-OK. Bioluminescence imaging showed that the size of the tumor formed by the xenografts infected with AdRIGF1R-OK was significantly smaller than the controls. Annexin V-FITC flow cytometry assay, immunofluorescence staining for cleaved Caspase3, and hoechst staining showed that more cells underwent apoptosis after infection with AdRIGF1R-OK. Luciferase reporter assay, crystal violet cell viability assay, and cell cycle analysis showed that the proliferation of melanoma cells infected with AdRIGF1R-OK was significantly decreased compared to the controls. This study demonstrates that the OK system is effective to silence gene expression, with promising potential to treat melanoma and other diseases.
Association between NER pathway gene polymorphisms and Wilms tumor risk Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-08 Jinhong Zhu, Wen Fu, Wei Jia, Huimin Xia, Guo-Chang Liu, Jing He
Nucleotide excision repair (NER) is an essential mechanism of the body in defending exogenous carcinogens-induced DNA damages. Defects in NER may impair DNA repair capacity, and thereby increase genome instability and cancer susceptibility. To explore genetic predispositions to Wilms tumor, we conducted a case-control study totaling 145 neuroblastoma cases and 531 healthy controls. We systematically selected 19 potentially functional single nucleotide polymorphisms (SNPs) in six key genes within the NER pathway (ERCC1, XPA, XPC, XPD, XPF, XPG). Odds ratio (OR) and 95% confidence interval (CI) were calculated to measure the strength of associations. We identified significant associations between two XPD SNPs and Wilms tumor risk. The XPDp rs3810366 olymorphism significantly enhanced Wilms tumor risk (dominant model: adjusted OR=2.12, 95% CI=1.26-3.57). Likewise, XPD rs238406 conferred significantly increased risk for the disease (dominant model: adjusted OR=2.30, 95% CI=1.40-3.80; recessive model: adjusted OR=1.64, 95% CI=1.11-2.44). Moreover, online expression quantitative trait locus (eQTL) analysis demonstrated that these two polymorphisms significantly affected XPD gene expression in transformed fibroblasts cells. Our study provided evidence of the association between the two XPD polymorphism and Wilms tumor risk. However, these findings warrant validation in larger studies.
Exposure of Endothelium to Biomimetic Flow Waveforms Yields Identification of miR-199a-5p as a Potent Regulator of Arteriogenesis Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-08 Joshua L. Heuslein, Catherine M. Gorick, Stephanie P. McDonnell, Ji Song, Brian H. Annex, Richard J. Price
RNAa and Vector-Mediated Overexpression of DIRAS1 Suppresses Tumor Growth and Migration in Renal Cell Carcinoma Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-08 Xin Xu, Jiangfeng Li, Song Wang, Xiangyi Zheng, Liping Xie
The downregulation of DIRAS1 has been suggested to potentially contribute to tumor development and progression in several human cancers. However, the role of DIRAS1 in renal cell carcinoma (RCC) remains elusive. In this study, we examined the DIRAS1 expression level in RCC cell lines and tissues. Both RNA activation (RNAa) and vector transfection methods were utilized to upregulate the expression of DIRAS1 in RCC cells. Expression analysis revealed that DIRAS1 was significantly downregulated in RCC cell lines and tissues compared with nontumorigenic renal cells and adjacent non-tumor tissues individually. Promoter methylation analysis indicated that the reduced DIRAS1 expression might be partly mediated by epigenetic modulation. The RNAa-mediated overexpression of DIRAS1 inhibited cell proliferation and tumorigenicity in vitro and in vivo. The re-activation of DIRAS1 also promoted apoptosis and suppressed migration and invasion in RCC cells. The ectopic expression of DIRAS1 via an expression vector recapitulated the RNAa results. These results reveal that DIRAS1 functioning as a putative tumor suppressor in RCC cells, could potentially be a therapeutic target, and RNAa could be a therapeutic strategy for RCC in future.
A transcriptome level study identifies changing expression profiles for ossification of ligamentum flavum of the spine Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-07 Yawei Han, Yuheng Hong, Liandong Li, Tengshuai Li, Zhen Zhang, Jingzhao Wang, Han Xia, Yutao Tang, Zhemin Shi, Xiaohui Han, Ting Chen, Qi Liu, Mengxia Zhang, Kun Zhang, Wei Hong, Yuan Xue
Ossification of the ligamentum flavum (OLF) is a common spinal disorder that causes myelopathy and radiculopathy. Non-coding RNAs (ncRNAs) are involved in numerous pathological processes, however, very few ncRNAs have been identified to be correlated with OLF. Here, we compared expressions of lncRNA, mRNA, circRNA and microRNA in ligamentum flavum tissues from OLF patients and healthy volunteers through mRNA, lncRNA and circRNA microarrays, and microRNA sequencing. A total of 2054 mRNAs, 2567 lncRNAs, 627 circRNAs and 28 miRNAs were altered during the process of OLF. Quantitative PCR confirmed the differential expressions of selected mRNAs and ncRNAs. lncRNA-mRNA co-expression network, miRNA-mRNA target prediction network and competing endogenous RNAs (ceRNAs) network of circRNA-miRNA-mRNA were constructed based on the correlation analysis of the differentially expressed RNA transcripts. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for the differentially expressed mRNAs and the miRNAs-predicted target genes were performed. In addition, a deregulated miRNA-19b-3p-based miRNA-circRNA-lncRNA-mRNA network was confirmed, by gain-of-function and loss-of-function experiments, to function in the process of ossification. Taken together, this study provides a systematic perspective on the potential function of ncRNAs in the pathogenesis of OLF.
BMP4 Upregulation Is Associated with Acquired Drug Resistance and Fatty Acid Metabolism in EGFR-Mutant Non-Small Cell Lung Cancer Cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-04 Duc-Hiep Bach, Thi-Thu-Trang Luu, Donghwa Kim, Yong Jin An, Hyen Joo Park, Sunghyouk Park, Sang Kook Lee
Lung cancer is the leading cause of cancer-associated deaths worldwide. In particular, non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR) mutations are associated with resistance development of EGFR tyrosine kinase inhibitors (EGFR-TKIs) treatment. Recent findings suggest that bone morphogenetic proteins (BMPs) and microRNAs (miRNAs) might act as oncogenes or tumor suppressors in the tumor microenvironment. In this study, for the first time, we identified the potential roles of BMPs and miRNAs involved in EGFR-TKI resistance by analyzing datasets from a pair of parental cells and NSCLC cells with acquired EGFR TKI-resistance. BMP4 was observed to be significantly over-expressed in the EGFR-TKI resistant cells, and its mechanism of action was strongly associated with the induction of cancer cell energy metabolism through the modulation of Acyl-CoA synthetase long-chain family member 4. In addition, miR-139-5p was observed to be significantly down-regulated in the resistant NSCLC cells. The combination of miR-139-5p and yuanhuadine, a naturally-derived antitumor agent, synergistically suppressed BMP4 expression in the resistant cells. We further confirmed that LDN-193189, a small molecule BMP receptor 1 inhibitor, effectively inhibited tumor growth in a xenograft nude mouse model implanted with the EFGR-TKI resistant cells. These findings suggest a novel role of BMP4-mediated tumorigenesis in the progression of acquired drug resistance in EGFR-mutant NSCLC cells.
Generation of Human Immunodeficiency Virus-resistant Macrophages from Induced Pluripotent Stem Cells by using Transcriptional Gene Silencing and Promoter-targeted RNA Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-04 Kei Higaki, Masako Hirao, Ai Kawana-Tachikawa, Shoichi Iriguchi, Ayako Kumagai, Norihiro Ueda, Wang Bo, Sanae Kamibayashi, Akira Watanabe, Hiromitsu Nakauchi, Kazuo Suzuki, Shin Kaneko
Highly active anti-retroviral therapy (HAART) has markedly prolonged the prognosis of HIV-1 patients. However, lifelong dependency on HAART is a continuing challenge, and an effective therapeutic is much desired. Recently, introduction of short hairpin RNA (shRNA) targeting the HIV-1 promoter was found to suppress HIV-1 replication via transcriptional gene silencing (TGS). The technology is expected to be applied with hemato-lymphopoietic cell transplantation of HIV patients to suppress HIV transcription in transplanted hemato-lymphopoietic cells. Combination of the TGS technology with new cell transplantation strategy with induced pluripotent stem cell (iPSC)-derived hemato-lymphopoietic cells might contribute to new gene therapy in the HIV field. In this study, we evaluated iPSC-derived macrophage functions and feasibility of TGS technology in macrophages. Human iPSCs were transduced with shRNAs targeting the HIV-1 promoter region (shPromA) by using a lentiviral vector. The shPromA-transfected iPSCs were successfully differentiated into functional macrophages, and they exhibited strong protection against HIV-1 replication with alteration in the histone structure of the HIV-1 promoter region to induce heterochromatin formation. These results indicated that iPS-derived macrophage is a useful tool to investigate HIV infection and protection, and that the TGS technology targeting the HIV promoter is a potential candidate of new gene therapy.
Molecular recognition and in vitro targeted inhibition of renal cell carcinoma using a DNA aptamer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-08-04 Hui Zhang, Zhibo Wang, Lin Xie, Yibin Zhang, Tanggang Deng, Jianglin Li, Jing Liu, Wei Xiong, Lei Zhang, Lin Zhang, Bo Peng, Leye He, Mao Ye, Xiaoxiao Hu, Weihong Tan
Renal cell carcinoma (RCC) is the most common malignant tumor of the urinary system and has a high frequency of local invasion and distant metastasis. Although multiple advances have been made in diagnosis and therapy of RCC, the vast majority of patients with metastatic RCC remain incurable. In this study, an aptamer named SW-4 against RCC 786-O cells was identified from a known sequence pool. The identified aptamer exhibited high binding affinity for target cells with dissociation constants in the nanomolar range. Binding analysis revealed that SW-4 only bound to RCC 786-O cells, but not human embryonic kidney 293T cells or human proximal tubular HK-2 cells, indicating that SW-4 has excellent binding selectivity. By sequence optimization, the 26-nucleotide truncated SW-4b demonstrated improved binding affinity and was internalized into target cells via caveolae-mediated endocytosis in a temperature-dependent manner. Furthermore, fluorescence imaging confirmed that SW-4b accumulated at tumor sites in 786-O xenograft nude mice models and specifically recognized clinical RCC tissues. Meanwhile, SW-4b inhibited proliferation of 786-O cells by arresting cell cycle progression at the S phase. Taken together, these results indicate that SW-4b is a potential candidate for development into a novel tool for diagnosis and targeted therapy of RCC.
LncRNA ZEB1-AS1 was suppressed by p53 for renal fibrosis in diabetic nephropathy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-31 Juan Wang, Jian pang, Huiling Li, Jie Long, Fang Fang, Junxiang Chen, Xuejin Zhu, Xudong Xiang, Dongshan Zhang
The role of p53 in renal fibrosis is still controversial, and its underlying mechanisms remain not clear. Here, we showed that the pharmacological inhibition and genetic deletion of p53 in proximal tubular cells can attenuate renal dysfunction, tubular epithelial disruption and interstitial fibrosis in db/db and STZ-induced diabetic nephrology (DN) mice. In human renal proximal tubule (HK-2) cells, inhibition of p53 by PIF reduced the high glucose (HG)-induced extracellular matrix (ECM) accumulation and reversed the inhibitory effect of HG on mRNA expression levels of LncRNA ZEB1-AS1 and ZEB1. Interestingly, our results demonstrated that both LncRNA ZEB1-AS1 and ZEB1 exhibited anti-fibrotic role, while ZEB1 is positively regulated by LncRNA ZEB1-AS1 during HG treatment. Mechanistically, Lnc ZEB1-AS1 bound directly to H3K4 methyltransferase MLL1, and promoted H3K4me3 histone modification on ZEB1 promoter, which was reduced by HG treatment. ChIp analysis indicated the binding of p53 to the promoter region of Lnc ZEB1-AS1. Furthermore, the findings were verified by the kidney biopsies samples from patients with DN. Taken altogether, our results suggest that p53 may be a therapeutic target for renal fibrosis in DN.
Expression analysis of MALAT1, GAS5, SRA and NEAT1 long non-coding RNAs in breast cancer tissues from young women and women over 45 years of age Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-31 Asghar Arshi, Fatemeh Sadat Sharifi, MiladKhoramian Ghahfarokhi, Zahra Faghih, Abbas Doosti, Sara Ostovari, ElhamMahmoudi Maymand, Mohammad Mahdi Ghahramani Seno
Introduction Breast cancer, as the most common cancer in women worldwide, represents about 30% of all cancers affecting women. Long non-coding RNAs (lncRNAs) have been implicated in the regulation of several biological processes and their dysregulation in cancer has well been documented. In order to investigate possible age-dependent variations in expression profiles of lncRNAs, we evaluated the expression levels of four lncRNAs, i.e. MALAT1, GAS5, SRA and NEAT1, in breast cancer (BC) samples obtained from young (<45 years) or older (>45 years) women. Methods Twenty three tumor samples and fifteen normal tissues were collected from BC patients. All tumor and normal samples were morphologically confirmed by a pathologist. RNA was extracted from the tissues and cDNAs were then synthesized. The lncRNAs expression levels were evaluated by RT-qPCR. The changes in the expression levels were determined using ΔΔCt method. Results Compared to normal tissues, BC tissues from both age groups (women under 45 years of age and women above 45 years of age) showed upregulation of MALAT1 (P=0.003/ P=0.0002) SRA (P=0.005/P=0.0002), NEAT1 (P=0.010/P=0.0002) and downregulation of GAS5 (P=0.0002/P=0.0005). Additionally, our analysis showed significant and direct correlation between the age and the expression levels of three of the four lncRNAs studied in this work. All four lncRNAs were overexpressed in both MDA-MB-231 and MCF7 cell lines (P=0.1000). Conclusion Our data shows that MALAT1, GAS5, SRA and NEAT1 lncRNAs are dysregulated in breast cancer samples. However, except for MALAT1, the expression levels of all of these lncRNAs were significantly lower in cancers developed in younger cases, where poorer prognosis is suggested. Of note, GAS5 reduced expression has been documented to correlate with tumor progression.
Silencing of circRNA.2837 plays a protective role in sciatic nerve injury by functioning as a sponge of the miR-34 family via regulating neuronal autophagy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-25 Zhi-bin Zhou, Yu-long Niu, Gao-xiang Huang, Jia-jia Lu, Aimin Chen, Lei Zhu
Circular RNAs (circRNAs) represent a class of non-coding RNAs that are involved in transcriptional and posttranscriptional gene expression regulation and associated with different kinds of human diseases. However, the characterization and function of circRNAs in peripheral nerve injuries remain elusive. Here, we established a rat sciatic nerve injury model and identified at least 4942 distinct circRNA candidates and a series of circRNAs that were differentially expressed in injured nerve tissues compared with matched normal tissues. We characterized one frequently down-regulated circRNA, circRNA.2837, and further investigated its function in sciatic nerve injury. We found that circRNA.2837 regulated autophagy in neurons in vitro and in vivo, and down-regulation of circRNA.2837 alleviated sciatic nerve injury via inducing autophagy in vivo. Mechanistically, knockdown of circRNA.2837 may protect neurons against neurological injury by acting as a sponge for members of miR-34 family. Our findings suggested that differentially expressed circRNAs were involved in the pathogenesis of sciatic nerve injury, and circRNAs exerted regulatory functions in sciatic nerve injury and might be used as potential targets in sciatic nerve injury therapy.
Splice-modulating oligonucleotide QR-110 restores CEP290 mRNA and function in human c.2991+1655A>G LCA10 models Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-23 Kalyan Dulla, Monica Aguila, Amelia Lane, Katarina Jovanovic, David A. Parfitt, Iris Schulkens, Hee Lam Chan, Iris Schmidt, Wouter Beumer, Lars Vorthoren, Rob W.J. Collin, Alejandro Garanto, Lonneke Duijkers, Anna Brugulat-Panes, Ma’ayan Semo, Anthony A. Vugler, Patricia Biasutto, Peter Adamson, Michael E. Cheetham
Leber congenital amaurosis 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wildtype mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic and safety properties make QR-110 a promising candidate for treating LCA10 and clinical development is currently ongoing.
Selection of metastatic breast cancer cell-specific aptamers for the capture of CTCs with a metastatic phenotype by Cell-SELEX Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-19 Wan-Ming Li, Lin-Lin Zhou, Min Zheng, Jin Fang
Circulating tumor cells (CTCs) have the potential to predict metastasis, and the capture of CTCs based on their surface markers is mostly applied for CTC detection. Considering that the CTCs with a metastatic phenotype preferably form a metastatic focus and that aptamers have the ability to bind targets with high specificity and affinity, we selected aptamers directed toward metastatic cells by subtractive Cell-SELEX technology using highly metastatic MDA-MB-231 cells as the target cell and low-metastatic MCF-7 cells as the negative cell for the capture of metastatic-CTCs. Affinity and selectivity assays showed that aptamer M3 had the highest affinity, with a Kd of 45.6 ± 1.2 nM, and had good specificity against several other types of metastatic cancer cells. Based on these findings, we developed an M3-based capture system for CTC enrichment, which have the capability to specifically capture the metastatic cells MDA-MB-231 mixed with non-metastatic MCF-7 cells and CTCs derived from metastatic breast cancer patient peripheral blood. A further comparative analysis with the anti-EpCAM probe showed that M3 probe captured epithelia feature-deletion metastatic cells. We developed an aptamer-based CTC capture system through the selection of aptamers by taking whole metastatic cells, not known molecules, as targets, which provided a new insight into CTC capture and Cell-SELEX application.
Intratracheal administration of siRNA dry powder targeting vascular endothelial growth factor inhibits lung tumor growth in mice Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-17 Kei Miwata, Hirokazu Okamoto, Taku Nakashima, Daisuke Ihara, Yasushi Horimasu, Takeshi Masuda, Shintaro Miyamoto, Hiroshi Iwamoto, Kazunori Fujitaka, Hironobu Hamada, Ayumi Shibata, Takaaki Ito, Tomoyuki Okuda, Noboru Hattori
Inhalation therapy using small-interfering RNA (siRNA) is a potentially effective therapeutic strategy for lung cancer because of its high gene silencing effects and sequence specificity. Previous studies reported that intratracheal administration of siRNA using pressurized metered dose inhalers or nebulizers could suppress tumor growth in murine lung metastatic models. Although dry powder inhalers are promising devices due to their low cost, good portability and preservability, the anti-tumor effects of siRNA dry powder have not been elucidated. To evaluate the gene silencing and anti-tumor effects of intratracheally delivered siRNA dry powder, vascular endothelial growth factor-specific siRNA (VEGF-siRNA) dry powder was administered intratracheally to mice with metastatic lung tumors consisting of B16F10 melanoma cells or Lewis lung carcinoma cells. A single intratracheal administration of VEGF-siRNA dry powder reduced VEGF levels in both bronchoalveolar lavage fluid and lung tumor tissue. Furthermore, repeated intratracheal administration of VEGF-siRNA dry powder suppressed the number of visible metastatic foci on the lung surface and tumor area in lung tissues. Taken together, intratracheal administration of siRNA dry powder could be a novel therapeutic strategy for lung cancer through the suppression of specific genes expressed in lung tumor tissue.
DROSHA knockout leads to enhancement of viral titers for vectors encoding miRNA adapted shRNAs (shRNAmiRs) Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-09 Hee Ho Park, Robinson Triboulet, Martin Bentler, Swaroopa Guda, Peng Du, Haiming Xu, Richard I. Gregory, Christian Brendel, David A. Williams
RNA interference (RNAi)-based gene therapy using miRNA adapted shRNAs (shRNAmiR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNAmiR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous Microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the Microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK 293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and Western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin containing Venus transcripts in DROSHA KO producer cells consistent with reduced Microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin containing constructs. We confirmed the absence of significant shRNAmiR processing by Northern blot analysis and showed that this correlated with an increase in the amount of full length vector genomic RNA. These findings may have important implications in future production of viral shRNAmiR-containing vectors for RNAi-based therapy.
Long non-coding RNA profiling reveals an abundant MDNCR that promotes differentiation of myoblasts via sponging miR-133a Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-09 Hui Li, Jiameng Yang, Rui Jiang, Xuefeng Wei, Chengchuang Song, Yongzhen Huang, Xianyong Lan, Chuzhao Lei, Yun Ma, Linyong Hu, Hong Chen
Muscle development is regulated under a series of complicate processes, and non-coding RNAs such as lncRNA has been reported to play important roles in regulating skeletal myogenesis and diseases. Here we profile the expression of lncRNA in cattle skeletal muscle tissue from fetus and adult developmental stages, and detect 13,580 lncRNA candidates. Many of these lncRNAs are differentially expressed between two developmental stages. We further characterize one abundant lncRNA—the highest expression level of all down-regulated lncRNA, which we named muscle differentiation-associated long non-coding RNA (MDNCR). Via luciferase screening, RNA binding protein immunoprecipitation (RIP) and RNA pulldown assays, MDNCR was observed to directly bind to miR-133a with thirty-two potential binding sites. GosB was identified as a target of miR-133a by luciferase activity, quantitative real-time PCR (qPCR) and western blotting assays. Overexpression of MDNCR increased the expression of GosB, while this effect was abolished by miR-133a. We found MDNCR to promote myoblasts differentiation and to inhibit cell proliferation by sponging miR-133a. These results demonstrated that MDNCR binding miR-133a promotes cell differentiation via targeting GosB in cattle primary myoblasts.
M6APred-EL: A sequence-based predictor for identifying N6-methyladenosine sites using ensemble learning Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-09 Leyi Wei, Huangrong Chen, Ran Su
N6-methyladenosine (m6A) modification is the most abundant RNA methylation modification that involves various biological progresses, such as RNA splicing and degradation. Recent studies have demonstrated the feasibility of identifying m6A-peaks using high-throughput sequencing techniques. However, the use of such techniques cannot accurately identify specific methylated sites, which are important for better understanding of m6A functions. In this study, we develop a novel machine learning based predictor called M6APred-EL for the identification of m6A sites. To predict m6A sites accurately within genomic sequences, we trained an ensemble of three support vector machine classifiers that explore the position-specific information and physical chemical information from position-specific k-mer nucleotide propensity, physical-chemical properties, and ring-function-hydrogen-chemical properties. We examined and compared the performance of our predictor with other state-of-the-art methods on benchmarking dataset. Comparative results showed that the proposed M6APred-EL performed more accurately for the m6A site identification. Moreover, a user-friendly webserver that implements the proposed M6APred-EL is well established and is currently available in http://server.malab.cn/M6APred-EL/. It is expected to be a practical and effective tool for the investigation of m6A functional mechanisms.
Novel mechanism of doxorubicin resistance and tumorigenesis mediated by microRNA-501-5p suppressed BLID Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-05 Yun-chao Xu, Xu Liu, Min Li, Yan Li, Chun-yan Li, Ying Lu, Jaceline Sanches, Lu Wang, Yue Du, Li-min Mao, Si-bo Zuo, Hui-ting Liu, Jie Shen, Bo Wang, Li Hou, Lian-hong Li, Jian-wu Tang, Jing-fang Ju, Bo Song
Doxorubicin is a widely used anthracycline-based anti-tumor agent for both solid and liquid tumors. Mounting evidences have demonstrated that microRNAs (miRNAs) are involved in the chemoresistance and tumorigenesis. However, the roles of microRNA-501-5p (miR-501) on the doxorubicin resistance and gastric cancer cell proliferation and invasion are still not fully understood. In this study, we identified that BLID (BH3-like motif containing protein, cell death inducer) was directly regulated by miR-501 at the post-transcriptional level in multiple gastric cancer cell lines. The endogenous miR-501 was higher, whereas BLID was lower in doxorubicin resistant gastric cancer SGC7901/ADR cells compared to its parental SGC7901 cells. miR-501 suppressed gastric cancer cell apoptosis and induced resistance to doxorubicin, and enhanced cell proliferation, migration and invasion. Subcutaneous injection of miR-501 lentivirus infected SGC7901 cells resulted in rapid growth of xenograft tumors and resistance to doxorubicin treatment than injection of SGC7901 cells. This is achieved at least partially by directly targeting BLID and subsequent inactivation of caspase-9, -3 and phosphorylation of Akt. Taken together, miR-501 induces doxorubicin resistance and enhances the tumorigenesis of gastric cancer cell via suppressing BLID. miR-501 might be a potential target for doxorubicin resistance and gastric cancer therapeutics.
Elevated tristetraprolin impairs trophoblast invasion in women with recurrent miscarriage by destabilization of HOTAIR Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-06 Fu-Ju Tian, Xiao-Ying He, Jie Wang, Xiao Li, Xiao-Ling Ma, Fan Wu, Jing Zhang, Xiao-Rui Liu, Xiao-Li Qin, Yan Zhang, Wei-Hong Zeng, Yi Lin
Tristetraprolin (TTP), regulates the stability of multiple targets that have important biological roles. However, the role of TTP in trophoblasts at the maternal-fetal interface remains poorly understood. We demonstrated that TTP was up-regulated in placental trophoblasts from patients with recurrent miscarriages (RM). Immunofluorescence and immunoblotting analyses indicated that TTP was redistributed from the nucleus to the cytoplasm in trophoblasts from patients with RM. Trophoblast invasion and proliferation was repressed by TTP overexpression, and was enhanced by TTP knockdown. Interestingly, TTP knockdown promoted trophoblast invasion in an ex vivo explant culture model. Furthermore, TTP overexpression in trophoblasts significantly inhibited the expression of the lncRNA HOTAIR. TTP was found to regulate HOTAIR expression by a post-transcriptional mechanism. To RNA immunoprecipitation (RIP) and RNA-protein pull-down identified TTP as a specific binding partner that decreased the half-life of HOTAIR and lowered steady-state HOTAIR expression levels, indicating a novel posttranscriptional regulatory mechanism. Our findings identify a novel function for TTP in lncRNA regulation, and provide important insights into the regulation of lncRNA expression. This study reveals a new pathway governing the regulation of TTP/HOTAIR in trophoblast cell invasion during early pregnancy.
Hepatocyte growth factor regulates miR-206-HDAC4 cascade to control the neurogenic muscle atrophy following surgical denervation in mice Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-06 Wooshik Choi, Junghun Lee, Jaeman Lee, Kyeong Ryang Ko, Sunyoung Kim
Hepatocyte growth factor (HGF) has been well characterized for its roles in the migration of muscle progenitors during embryogenesis and the differentiation of muscle stem cells, yet its function(s) in adult neurogenic muscle atrophic conditions is poorly understood. Here, we investigated whether HGF/c-met signaling has any effects on muscle atrophic conditions. It was found that HGF expression was upregulated in skeletal muscle tissue following surgical denervation and in hSOD1-G93A transgenic mice showing severe muscle loss. Pharmacological inhibition of c-met receptor decreased the expression level of pri-miR-206, enhanced that of HDAC4 and atrogenes, and resulted in increased muscle atrophy. In C2C12 cells, HGF inhibited phosphorylation of Smad3, relieved TGF-β-mediated suppression of miR-206 expression via JNK. When extra HGF was exogenously provided through intramuscular injection of plasmid DNA expressing HGF, the extent of muscle atrophy was reduced, and the levels of all affected biochemical markers were changed accordingly including those of primary and mature miR-206, HDAC4, and various atrogenes. Taken together, our finding suggested that HGF might play an important role(s) in regard to neurogenic muscle atrophy and that HGF might be used as a platform to develop therapeutics for neuromuscular disorders.
A novel lncRNA, LINC00460 affects cell proliferation and apoptosis by regulating KLF2 and CUL4A expression in colorectal cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-07-06 Yifan Lian, Changsheng Yan, Hongzhi Xu, Jiebin Yang, Yang Yu, Jing zhou, Yongguo Shi, Jianlin Ren, Guozhong Ji, Keming Wang
Emerging evidence has proven that long noncoding RNAs (lncRNAs) play important roles in human colorectal cancer (CRC) biology, while few lncRNAs have been characterized in CRC. Therefore, the functional significance of lncRNAs in the malignant progression of CRC still needs to be further explored. In this study, through analysing TCGA RNA sequencing data and other publicly available microarray data, we found that a novel lncRNA, LINC00460, whose expression was significantly up-regulated in CRC tissues compared to adjacent normal tissues. Consistently, qRT-PCR results also verified that LINC00460 was over-expressed in CRC tissues and cells. Furthermore, high LINC00460 expression levels in CRC specimens were correlated with larger tumor size, advanced tumor stage, lymph node metastasis and shorter overall survival. In vitro and in vivo assays of LINC00460 alterations revealed a complex integrated phenotype affecting cell growth and apoptosis. Mechanistically, LINC00460 repressed kruppel like factor 2 (KLF2) transcription by binding to enhancer of zeste homolog 2 (EZH2). LINC00460 also functioned as a molecular sponge for miR-149-5p, antagonizing its ability to repress cullin 4A (CUL4A) protein translation. Taken together, our findings support a model in which the LINC00460/EZH2/KLF2 and LINC00460/miR-149-5p/CUL4A crosstalk serve as critical effectors in CRC tumorigenesis and progression, suggesting new therapeutic directions in colorectal cancer.
Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity Without HPLC Purification Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-30 Sriram Vaidyanathan, Krist T. Azizian, AKM Ashiqul Haque, Jordana M. Henderson, Ayal Hendel, Sabrina Shore, Justin S. Antony, Richard I. Hogrefe, Michael S.D. Kormann, Matthew H. Porteus, Anton P. McCaffrey
Reaching for the Stars in the Brain: Polymer-Mediated Gene Delivery to Human Astrocytes Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-28 Chaitanya R. Joshi, Vijay Raghavan, Sivakumar Vijayaraghavalu, Yue Gao, Manju Saraswathy, Vinod Labhasetwar, Anuja Ghorpade
Astrocytes, the ‘star-shaped’ glial cells, are appealing gene delivery targets to treat neurological diseases due to their diverse roles in brain homeostasis and disease. Cationic polymers have successfully delivered genes to mammalian cells, hence present a viable, non-immunogenic alternative to widely used viral vectors. In this study, we investigated the gene delivery potential of a series of arginine- and polyethylene glycol-modified, siloxane-based polyethylenimine analogues in primary cultured human neural cells (neurons and astrocytes) and in mice. Plasmid DNAs encoding luciferase reporter were used to measure gene expression. We hypothesized that polyplexes with arginine would help in cellular transport of the DNA, including across the blood-brain barrier, polyethylene glycol will stabilize polyethylenimine and reduce its toxicity; while maintaining its DNA condensing ability. Polyplexes were non-toxic to human neural cells and red blood cells. Cellular uptake of polyplexes and sustained gene expression was seen in human astrocytes as well as in mouse brains post-intravenous injections. The polyplexes also delivered and expressed genes driven by astrocyte-restricted glial fibrillary acidic protein promoters, which are weaker than viral promoters. To our knowledge, the presented work validates a biocompatible and effective polymer-facilitated gene delivery system for both human brain cells and mice for the first time.
Recurrence-associated long non-coding RNA signature for determining the risk of recurrence in patients with colon cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-26 Meng Zhou, Long Hu, Zicheng Zhang, Nan Wu, Jie Sun, Jianzhong Su
Patients with colon cancer often faced high risk of disease recurrence within 5 years of treatment that is the major cause of cancer mortality. Reliable molecular markers were required to improve most effective personalized therapy. Here, we identified a recurrence-associated six-lncRNA signature (LINC0184, AC105243.1, LOC101928168, ILF3-AS1, MIR31HG and AC006329.1) that can effectively distinguish between high and low risk of cancer recurrence form 389 patients of discovery dataset, and validated its robust performance in four independent datasets comprising a total of 906 colon cancer patients. We found that the six-lncRNA signature was an independent predictive factor of disease recurrence in multivariate analysis, and was superior to the performance of clinical factors and known gene signature. Furthermore, in silico functional analysis showed that the six-lncRNA signature associated coding genes are significantly enriched in proliferation and angiogenesis, cell death, as well as critical cancer pathways that could play important roles in colon cancer recurrence. Together, the six-lncRNA signature holds great potential for recurrence risk assessment and personalized management of colon cancer patients.
Ex vivo COL7A1 correction for Recessive Dystrophic Epidermolysis Bullosa using CRISPR/Cas9 and Homology Directed Repair Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-26 Araksya Izmiryan, Clarisse Ganier, Matteo Bovolenta, Alain Schmitt, Fulvio Mavilio, Alain Hovnanian
Recessive Dystrophic Epidermolysis Bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. RDEB is caused by a wide variety of mutations in COL7A1 encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-ddPCR resulting in 11 and 15.7% of corrected COL7A1 mRNA expression, respectively without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.
Impact of miRNA-mRNA profiling and their correlation on medulloblastoma tumorigenesis Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-19 Vinod Kumar, Virender Kumar, Amit Kumar Chaudhary, Donald W. Coulter, Timothy McGuire, Ram I. Mahato
Medulloblastoma (MB) is a clinically challenging, childhood brain tumor with diverse genetic makeup and differential miRNA profile. Aiming to identify deregulated miRNAs in MB, miRNA expression profile of human MB samples was compared to normal cerebellar tissues. As a result, 8 upregulated and 64 downregulated miRNAs were identified in MB samples. Although various algorithms have been developed to predict the interaction between miRNA-mRNA pairs, the complexity and fidelity of miRNA-mRNA remain a concern. Therefore, to identify the signatures of miRNA-mRNA interactions essential for MB pathogenesis, miRNA profiling, RNA-sequencing, and ingenuity pathway analysis (IPA) were performed in the same primary human MB samples. Further, when miR-217 was inhibited, a significant upregulation of predicted target genes SIRT1, ROBO1, FOXO3, and SMAD7 in HDMB03 cells was observed confirming the validity of our approach. Functional analysis revealed that inhibition of miR-217 in HDMB03 cells suppresses colony formation, migration, invasion promoted apoptosis, and arrested cell population in S phase, indicating that manipulation of miR-217 may have a therapeutic potential for MB patients. Therefore, our study provides an essential platform for future investigations of specific miRNAs responsible for MB pathogenesis.
Long-term morpholino oligomers in hexose elicits long-lasting therapeutic improvements in mdx mice Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-21 Gang Han, Caorui Lin, Hanhan Ning, Xianjun Gao, HaiFang Yin
Approval of antisense oligonucleotide eteplirsen highlights the promise of exon- skipping therapeutics for Duchenne muscular dystrophy patients. However, the limited efficacy of eteplirsen underscores the importance to improve systemic delivery and efficacy. Recently we demonstrated that a glucose-fructose (GF) delivery formulation effectively potentiates phosphorodiamidate morpholino oligomer (PMO). Considering the clinical potential of GF, it is important to determine the long-term compatibility and efficacy with PMO in mdx mice prior to clinical translation. Here, we report that yearlong administration of a clinically applicable PMO dose (50mg/kg/week for 3 weeks followed by 50mg/kg/month for 11 months) with GF elicited sustainably high levels of dystrophin expression in mdx mice, with up to 45% of normal level of dystrophin restored in most peripheral muscles without any detectable toxicity. Importantly, PMO-GF resulted in phenotypical rescue and mitochondrial biogenesis with functional improvement. Carbohydrate metabolites measurements revealed improved metabolic and energetic conditions after PMO-GF treatment in mdx mice without metabolic anomaly. Collectively, our study shows PMO-GF’s ability to elicit long-lasting therapeutic effects with tolerable toxicity and represents a new treatment modality for Duchenne muscular dystrophy, and provides guidelines for antisense oligonucleotides with GF in clinical use.
PAK1 promotes the proliferation and inhibits the apoptosis of human spermatogonial stem cells through PDK1/KDR/ZNF367 and ERK1/2 and AKT pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-21 Hongyong Fu, Wenhui Zhang, Qingqing Yuan, Minghui Niu, Fan Zhou, Qianqian Qiu, Guoping Mao, Hong Wang, Liping Wen, Min Sun, Zheng Li, Zuping He
Spermatogonial stem cells (SSCs) have significant applications in reproductive and regenerative medicine. However, nothing is known about genes in mediating human SSCs. Here we have explored for the first time the function and mechanism of PAK1 in regulating proliferation and apoptosis of human SSC line. PAK1 level was up-regulated by EGF but not GDNF or FGF2. PAK1 promoted proliferation and DNA synthesis of human SSC line, whereas it suppressed their apoptosis in vitro and in vivo. RNA sequencing identified that PDK1, ZNF367 and KDR levels were downregulated by PAK1 knockdown. Immunoprecipitation and Western blots demonstrated that PAK1 interacted with PDK1. PDK1 and KDR levels were decreased by ZNF367-siRNAs. The proliferation of human SSC line was reduced by PDK1-, KDR- and ZNF367-siRNAs, whereas their apoptosis was enhanced by these siRNAs. The levels of phos-ERK1/2, phos-AKT and cyclin A were decreased by PAK1 siRNAs. Tissue arrays showed that PAK1 level was lower in non-obstructive azoospermia patients. Collectively, PAK1 was identified as the first molecule that controls proliferation and apoptosis of human SSC line through PDK1/KDR/ZNF367 and ERK1/2 and AKT pathway. This study provides novel gene regulation and networks underlying the fate of human SSCs and offers new molecular targets for human SSCs in translational medicine.
miR-7 modulates human embryonic stem cell differentiation into insulin-producing beta-like cells and contributes to cell maturation Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-15 Javier López-Beas, Vivian Capilla-González, Yolanda Aguilera, Nuria Mellado, Christian C. Lachaud, Franz Martín, Tarik Smani, Bernat Soria, Abdelkrim Hmadcha
Human pluripotent stem cells retain the extraordinary capacity to differentiate into pancreatic beta cells. For this particular lineage, more effort is still required to stress the importance of developing an efficient, reproducible, easy and cost-effective differentiation protocol to obtain more mature, homogenous and functional insulin-secreting cells. In addition, microRNAs (miRNAs or miRs) have emerged as a class of small non-coding RNAs that regulate many cellular processes, including pancreatic differentiation. Some miRNAs are known to be preferentially expressed in islets. Of note, miR-375 and miR-7 are two of the most abundant pancreatic miRNAs and are necessary for proper pancreatic islet development. Here, we provide new insight into specific miRNAs involved in pancreatic differentiation. We found that miR-7 is differentially expressed during the differentiation of human embryonic stem cells (hESCs) into a beta cell-like phenotype and that its modulation plays an important role in generating mature pancreatic beta cells. This strategy may be exploited to optimize the potential for in vitro differentiation of hESCs into insulin-producing beta-like cells for use in preclinical studies and future clinical applications as well as the prospective uses of miRNAs to improve this process.
Gemcitabine-incorporated G-quadruplex aptamer for targeted drug delivery into pancreas cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-06-15 Jun Young Park, Ye Lim Cho, Ju Ri Chae, Sung Hwan Moon, Won Gil Cho, Yun Jung Choi, Soo Jin Lee, Won Jun Kang
RNA polymerase II activity of type 3 Pol III promoters Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-08 Zongliang Gao, Elena Herrera-Carrillo, Ben Berkhout
In eukaryotes, three RNA polymerases (Pol I, II and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites and are therefore widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters (7SK, U6 and H1) mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1>>U6>7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nucleotides upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters and offer additional flexibility for biotechnology applications of these genetic elements.
Circulating miRNAs as predictive biomarkers of type 2 diabetes mellitus development in coronary heart disease patients from the CORDIOPREV study Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-08 Rosa Jimenez-Lucena, Oriol Alberto Rangel-Zúñiga, Juan Francisco Alcalá-Díaz, Javier Lopez-Moreno, Irene Roncero-Ramos, Helena Molina-Abril, Elena Yubero-Serrano, Javier Caballero-Villarraso, Javier Delgado-Lista, Justo Pastor Castaño, Jose Maria Ordovás, Pablo Pérez-Martinez, Antonio Camargo, Jose Lopez-Miranda
Circulating miRNAs have been proposed as type 2 diabetes biomarkers, and may be a more sensitive way to predict development of the disease than the currently used tools. Our aim was to identify whether circulating miRNAs, added to clinical and biochemical markers, could have better potential predicting type 2 diabetes. The study included 462 non-diabetic patients at baseline in the CORDIOPREV study. After a median follow-up of 60 months, 107 of them developed type 2 diabetes. Plasma levels of 24 miRNAs were measured at baseline by qRT-PCR and other strong biomarkers to predict diabetes were determined. The ROC-analysis identified 9 miRNAs, which added to HbA1c, have a greater predictive value in early diagnosis of type 2 diabetes (AUC = 0.8342) than HbA1c alone (AUC = 0.6950). The miRNAs and HbA1c based model did not improve when the FINDRISC was included (AUC=0.8293). Cox-regression analyses showed that patients with low miR-103, miR-28-3p, miR-29a, miR-9 and high miR-30a-5p and miR-150 circulating levels have higher risk of disease (HR= 11.27; 95% CI: 2.61 – 48.65). Our results suggest that circulating miRNAs could potentially be used as a new tool for predicting the development of type 2 diabetes in clinical practice.
Self-amplifying Replicon RNA delivery to Dendritic Cells by Cationic Lipids Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-04 Pavlos C. Englezou, Cedric Sapet, Thomas Démoulins, Panagiota Milona, Thomas Ebensen, Kai Schulze, Carlos-Alberto Guzman, Florent Poulhes, Olivier Zelphati, Nicolas Ruggli, Kenneth C. McCullough
Advances in RNA technology during the past two decades have led to the construction of replication-competent RNA termed replicons, RepRNA or self-amplifying mRNA with high potential for vaccine applications. Cytosolic delivery is essential for their translation and self-replication - without infectious progeny generation - providing high levels of antigen expression for inducing humoral and cellular immunity. Synthetic nanoparticle-based delivery vehicles can both protect the RNA molecules and facilitate targeting of dendritic cells – critical for immune defence development. Several cationic lipids were assessed with RepRNA generated from classical swine fever virus encoding nucleoprotein genes of influenza A virus. The non-cytopathogenic nature of the RNA allowed targeting to dendritic cells without destroying the cells – important for prolonged antigen production and presentation. Certain lipids were more effective at delivery and at promoting translation of RepRNA than others. Selection of particular lipids provided delivery to dendritic cells that did result in translation, demonstrating that delivery efficiency could not guarantee translation. The observed translation in vitro was reproduced in vivo by inducing immune responses against the encoded influenza virus antigens. Cationic lipid-mediated delivery shows potential for promoting RepRNA vaccine delivery to dendritic cells, particularly if combined with additional delivery elements.
Parallel genome-wide profiling of coding and non-coding RNAs to identify novel regulatory components in embryonic heart development and maturated heart Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-04 Davood Sabour, Rui S.R. Machado, Jose P. Pinto, Susan Rohani, Raja Ghazanfar Ali Sahito, Jürgen Hescheler, Matthias E. Futschik, Agapios Sachinidis
Heart development is a complex process, tightly regulated by numerous molecular mechanisms. Key components of the regulatory network underlying heart development are transcription factors (TFs) and microRNAs (miRNAs), yet limited investigation of the role of miRNAs in heart development has taken place. Here, we report the first parallel genome-wide profiling of polyadenylated RNAs and miRNAs in a developing murine heart. These data enable us to identify dynamic activation or repression of numerous biological processes and signaling pathways. More than 200 miRNAs and 25 long non-coding RNAs were differentially expressed during embryonic heart development compared to the mature heart; most of these had not been previously associated with cardiogenesis. Integrative analysis of expression data and potential regulatory interactions suggested 28 miRNAs as novel regulators of embryonic heart development representing a considerable expansion of the current repertoire of known cardiac miRNAs. To facilitate follow-up investigations, we constructed HeartMiR (http://heartmir.sysbiolab.eu), an open access database and interactive visualization tool for the study of gene regulation by miRNAs during heart development.
MicroRNA-125b promotes hepatic stellate cell activation and liver fibrosis by activating RhoA signaling Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-03 Kai You, Song-Yang Li, Jiao Gong, Jian-Hong Fang, Chong Zhang, Min Zhang, Yunfei Yuan, Jine Yang, Shi-Mei Zhuang
MiR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs but not in hepatocytes during hepatic fibrogenesis in vivo and upon culture-activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor beta (TGF-β)-induced α-SMA expression and cell contraction of immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl4-treated mice. Mechanistically, overexpression of miR-125b in HSC enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSC and highlight its importance in hepatic fibrosis.
Translating SOD1 gene silencing towards the clinic: A highly efficacious, off-target free and biomarker-supported strategy for familial ALS Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-03 T. Iannitti, J.M. Scarrott, S. Likhite, I.R.P. Coldicott, K.E. Lewis, P.R. Heath, A. Higginbottom, M.A. Myszczynska, M. Milo, G.M. Hautbergue, K. Meyer, B.K. Kaspar, L. Ferraiuolo, P.J. Shaw, M. Azzouz
Twenty per cent of familial amyotrophic lateral sclerosis (fALS) cases are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of interfering RNA (RNAi) for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88% and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials.
Targeting the IGF1R pathway in breast cancer using antisense lncRNA-mediated promoter cis competition Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-03 Lingling Pian, Xue Wen, Lihua Kang, Zhaozhi Li, Yuanyuan Nie, Zhonghua Du, Dehai Yu, Lei Zhou, Lin Jia, Naifei Chen, Dan li, Songling Zhang, Wei Li, Andrew R. Hoffman, Jingnan Sun, Jiuwei Cui, Ji-Fan Hu
Aberrant insulin-like growth factor I receptor (IGF1R) signaling pathway serves as a well-established target for cancer drug therapy. The intragenic antisense long noncoding RNA (lncRNA) IRAIN, a putative tumor suppressor, is downregulated in breast cancer cells, while IGF1R is overexpressed, leading to an abnormal IGF1R/IRAIN ratio that promotes tumor growth. To precisely target this pathway, we developed an “antisense lncRNA-mediated intragenic cis competition” (ALIC) approach to therapeutically correct the elevated IGF1R/IRAIN bias in breast cancer cells. We used CRISPR-Cas9 gene editing to target the weak promoter of IRAIN antisense lncRNA and showed that in targeted clones, intragenic activation of the antisense lncRNA potently competed in cis with the promoter of the IGF1R sense mRNA. Notably, the normalization of IGF1R/IRAIN transcription inhibited the IGF1R signaling pathway in breast cancer cells, decreasing cell proliferation, tumor sphere formation, migration, and invasion. Using “nuclear RNA reverse transcription-associated trap” sequencing, we uncovered an IRAIN lncRNA-specific interactome containing gene targets involved in cell metastasis, signaling pathways, and cell immortalization. These data suggest that aberrantly upregulated IGF1R in breast cancer cells can be precisely targeted by cis transcription competition, thus providing a useful strategy to target disease genes in the development of novel precision medicine therapies.
Regulated zinc finger nucleases (ZFNs) by HIV-1 Tat to excise HIV-1 from host genome in infected and latently infected cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-03 Haiyan Ji, Panpan Lu, Baochi Liu, Xiying Qu, Yanan Wang, Zhengtao Jiang, Xinyi Yang, Yangcheng Zhong, He Yang, Hanyu Pan, Lin Zhao, Jianqing Xu, Hongzhou Lu, Huanzhang Zhu
Highly active anti-retroviral therapy (HAART) cannot clear the infected cells harboring HIV-1 proviral DNA from the HIV-1 infected patients. We previously demonstrated that Zinc-finger nucleases (ZFNs) could specifically and efficiently excise HIV-1 proviral DNA from the latently infected human T cells by targeting the long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent the unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFNs expression can be activated by the HIV-1 Tat protein. Our results show that functional expression of ZFNs induced by Tat excised the integrated proviral DNA of HIV-NL4-3-eGFP in approximate 30% population of HIV-1 infected cells. Meanwhile, the results from HIV-1 infected human primary T cells and latently infected T cells treated with the regulated ZFNs further validated proviral DNA could be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of the genome-editing technology for eradicating the HIV-1 proviral DNA from the infected host cells.
Thermoresponsive bacteriophage nanocarrier as a gene delivery vector targeted to the gastrointestinal tract Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-01 Katawut Namdee, Mattaka Khongkow, Suwimon Boonrungsiman, Naiyaphat Nittayasut, Paladd Asavarut, Sasithon Temisak, Nattika Saengkrit, Satit Puttipipatkhachorn, Amin Hajitou, Kiat Ruxrungtham, Teerapong Yata
The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine–glycine–aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract.
Stable transcriptional repression and parasitism of HIV-1 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-05-01 Surya Shrivastava, Paige Charlins, Amanda Ackley, Heather Embree, Boro Dropulic, Ramesh Akkina, Marc S. Weinberg, Kevin V. Morris
Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1; both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T-cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1 leading ultimately to a functional cure.
Increased frataxin expression induced in Friedreich ataxia cells by platinum TALE-VP64s or platinum TALE-SunTag Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-27 Khadija Cherif, Catherine Gérard, Joël Rousseau, Dominique L. Ouellet, Pierre Chapdelaine, Jacques P. Tremblay
Frataxin gene (FXN) expression is reduced in Friedreich's ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondria’s iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA), such as VP64 or P300, were used to increase the expression of that gene. Many effectors plTALEVP64, plTALEp300 and plTALESunTag targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted to select 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19 folds in different FRDA primary fibroblasts. Adeno-Associated Viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies in vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10 folds and in skeletal muscles by up to 35 folds. The aconitase activity is positively modulated by the frataxin level in mitochondria and was thus increased in vitro and in vivo by the increased frataxin expression.
Sp1 Suppresses MiR-3178 to Promote Metastasis Invasion Cascade via Upregulation of TRIOBP Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-26 Hui Wang, Kai Li, Yu Mei, Xuemei Huang, Zhenglin Li, Qingzhu Yang, Huanjie Yang
Specificity protein (Sp1) plays an important role in invasion-metastasis cascade. Sp1 regulation on protein coding genes have been extensively investigated, however little is known about its regulation on protein non-coding genes. In this study, miR-3178 is reported as a novel target of Sp1 in multiple cancer cell models. Sp1 functions as its transcriptional suppressor as evidenced by luciferase reporter and ChIP assays. In line with the pro-metastatic role of Sp1, miR-3178 exerts anti-metastasis function. Overexpression of miR-3178 inhibits both migration and invasion of highly metastatic prostate, lung and breast cancer cells while antagonizing miR-3178 promotes those events in their lowly metastatic counterparts. In vivo study demonstrates that miR-3178 suppresses the tail vein inoculated prostate cancer cells to form colonies in lung, lymph node and liver of BALB/c nude mice. MiR-3178 directly targets the 3' UTR of TRIOBP-1 and TRIOBP-5, the two isoforms of TRIOBP expressed in prostate, lung and breast cancer cells. Furthermore, overexpression of TRIOBP-1 could rescue miR-3178 inhibition on cell migration and invasion. Collectively, our findings reveal the regulatory axis of Sp1/miR-3178/TRIOBP in metastasis cascade. Our results suggest miR-3178 as a promising application to suppress metastasis in Sp1 overexpressed cancers.
The ASH1-miR-375-YWHAZ signaling axis regulates tumor properties in hepatocellular carcinoma Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-25 Juan-Feng Zhao, Qiu Zhao, Hui Hu, Jia-Zhi Liao, Ju-Sheng Lin, Chao Xia, Ying Chang, Jing Liu, An-Yuan Guo, Xing-Xing He
Hepatocellular carcinoma (HCC) is a worldwide malignance and the underlying mechanisms of this disease are not fully elucidated. In this study, the existence and function of ASH1-miR-375-YWHAZ signaling axis in HCC were determined. Our experiments and TCGA sequencing data analyses showed that ASH1 and miR-375 were significantly down-regulated, while YWHAZ was significantly up-regulated in HCC. Furthermore, we found that ASH1 positively regulates miR-375, and miR-375 directly down-regulates its target YWHAZ. Gain- and loss-of-function study demonstrated ASH1 and miR-375 function as tumor suppressors, while YWHAZ acts as an oncogene in HCC. Animal experiment indicated that si-YWHAZ delivered by nanoliposomes could suppress the growth of hepatoma xenografts and was well tolerant by nude mice. Further studies revealed that YWHAZ was involved in several protein networks such as cell autophagy, EMT, apoptosis, cell cycle, invasion and migration. In addition, the patient group with ASH1-high-expression-miR-375-high-expression-YWHAZ-low-expression was correlated with a better clinical prognosis compared with the opposite expression group. In conclusion, we proved the existence of ASH1-miR-375-YWHAZ signaling axis and interpreted its important role in driving HCC tumor progression.
miR-24 and miR-122 negatively regulate transforming growth factor-β/Smad signaling pathway in skeletal muscle fibrosis Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-22 Yaying Sun, Hui Wang, Yan Li, Shaohua Liu, Jiwu Chen, Hao Ying
Fibrosis is common after skeletal muscle injury, undermining tissue regeneration and function. The mechanism underlying skeletal muscle fibrosis remains unveiled. Transforming growth factor-β/Smad signaling pathway is supposed to play a pivotal role. However, how microRNAs interact with transforming growth factor-β/Smad related muscle fibrosis remains unclear. We showed that miR-24-3p and miR-122-5p declined in skeletal muscle fibrosis, which was a consequence of transforming growth factor-β. Up-regulating Smad4 suppressed two microRNAs, while inhibiting Smad4 elevated microRNAs. Luciferase reporter assay and chromatin immunoprecipitation confirmed that Smad4 directly inhibited two microRNAs. On the other hand, overexpression of these two miRs retarded fibrotic process. We further identified that Smad2 was a direct target of miR-24-3p, while miR-122-5p targeted transforming growth factor-β receptor-II. Both targets were important participants in transforming growth factor-β/Smad signaling. Taken together, a positive feedback loop in transforming growth factor-β/Smad4 signaling pathway in skeletal muscle fibrosis was identified. Transforming growth factor-β/Smad axis could be down-regulated by microRNAs. This effect, however, was suppressed by Smad4, the downstream of transforming growth factor-β.
Antibody-antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-19 Amy E. Arnold, Elise Malek-Adamian, Phuong U. Le, Anika Meng, Saúl Martínez-Montero, Kevin Petrecca, Masad J. Damha, Molly S. Shoichet
Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC invasion. We developed antibody-antisense oligonucleotide conjugates to target and reduce DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the glioblastoma stem cells, such as CD44 and EphA2, conjugated to chemically modified antisense oligonucleotides (AONs) against DRR/FAM107A, which were designed as chimeras of DNA and 2ʹ-deoxy-2ʹ-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize, accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first example of an antibody-antisense strategy against cancer stem cells.
Promoter-operating targeted expression of gene therapy in cancer: current stage and prospect Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-12 Chao Chen, Dongxu Yue, Liangyu Lei, Hairong Wang, Jia Lu, Ya Zhou, Shiming Liu, Tao Ding, Mengmeng Guo, Lin Xu
The technique of targeted expression of interesting genes, including distinct delivery systems and specific gene promoter-operating expression, is an important strategy for gene therapy against cancers. Up to now, extensive literatures documented the efficacy of distinct delivery systems, such as liposome system, nano-partical system and PEI system, and so on, in cancer gene therapy. However, related document on the potential value of using specific gene promoter, such as tumor suppressor, in cancer gene therapy was still scare. The main obstacle might be the selection of ideal gene promoter to operate interesting gene expression in cancer gene therapy is still not fully understood. Therefore, many efforts need to be done in order to make it a real power tool for the human clinical treatment for cancer patients. The purpose of this review is to clarify the current state and some problematics on development of promoter-operating targeted expression of interesting genes and highlight its potential in cancer gene therapy.
miR-199a-3p modulates MTOR and PAK4 pathways and inhibits tumor growth in a hepatocellular carcinoma transgenic mouse model Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-04-12 Elisa Callegari, Lucilla D’Abundo, Paola Guerriero, Carolina Simioni, Bahaeldin K. Elamin, Marta Russo, Alice Cani, Cristian Bassi, Barbara Zagatti, Luciano Giacomelli, Stella Blandamura, Farzaneh Moshiri, Simona Ultimo, Antonio Frassoldati, Giuseppe Altavilla, Laura Gramantieri, Luca Maria Neri, Silvia Sabbioni, Massimo Negrini
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Prognosis is poor, and therapeutic options are limited. microRNAs (miRNAs) have emerged as potential therapeutic molecules against cancer. Here, we investigated the therapeutic efficacy of miR-199a-3p, a miRNA highly expressed in normal liver and downregulated in virtually all HCCs. The therapeutic value of miR-199a-3p mimic molecules was assayed in the TG221 mouse, a transgenic model highly predisposed to the development of liver cancer. Administration of miR-199a-3p mimics in the TG221 transgenic mouse showing liver cancer led to a significant reduction of number and size of tumor nodules compared to control animals. In vivo delivery confirmed protein downregulation of the miR-199a-3p direct targets, mechanistic target of rapamycin (MTOR) and p21 activated kinase 4 (PAK4), ultimately leading to the repression of FOXM1. Remarkably, the anti-tumor activity of miR-199a-3p mimics was comparable to that obtained with sorafenib. These results suggested that miR-199a-3p may be considered a promising HCC therapeutic option.
De novo synthesis of elastin by exogenous delivery of synthetic modified messenger RNA into skin and elastin deficient cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-30 Mario Lescan, Regine Perl, Sonia Golombek, Martin Pilz, Ludmilla Hann, Mahua Yasmin, Andreas Behring, Timea Keller, Andrea Nolte-Karayel, Franziska Gruhn, Efrat Kochba, Yotam Levin, Christian Schlensak, Hans Peter Wendel, Meltem Avci-Adali
Elastin is one of the most important and abundant extracellular matrix (ECM) protein that provides elasticity and resilience to tissues and organs, including vascular walls, ligaments, skin, and lung. Besides hereditary diseases, such as Williams-Beuren-Syndrome (WBS), which results in reduced elastin synthesis, injuries, aging, or acquired diseases can lead to the degradation of existing elastin fibers. Thus, the de novo synthesis of elastin is required in several medical conditions to restore the elasticity of affected tissues. Here, we applied synthetic modified messenger RNA (mRNA) encoding tropoelastin (TE) for the de novo synthesis of elastin and determined the mRNA-mediated elastin synthesis in cells as well as ex vivo in porcine skin. EA.hy926 cells, human fibroblasts, and mesenchymal stem cells (MSCs) isolated from a patient with WBS were transfected with 2.5 μg TE mRNA. After 24 h, the production of elastin was analyzed by Fastin assay and dot blot analyses. Compared to untreated cells, significantly enhanced elastin amounts were detected in TE mRNA transfected cells. The delivered synthetic TE mRNA was even able to significantly increase the elastin production in elastin deficient MSCs. In porcine skin, approximately 20% higher elastin amount was detected after the intradermal delivery of synthetic mRNA by microinjection. In this study, we demonstrated the successful applicability of synthetic TE encoding mRNA to produce elastin in elastin-deficient cells as well as in skin. Thus, this auspicious mRNA-based integration-free method has a huge potential in the field of regenerative medicine to induce de novo elastin synthesis, e.g. in skin, blood vessels, or alveoli.
iRNA-3typeA: identifying 3-types of modification at RNA’s adenosine sites Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-30 Wei Chen, Pengmian Feng, Hui Yang, Hui Ding, Hao Lin, Kuo-Chen Chou
RNA modifications are referred to the addition of chemical groups to nucleotides, or their local structural changes. Knowledge about the occurrence sites of these modifications is essential for in-depth understanding the biological functions and mechanisms, and treating some genomic diseases as well. With the avalanche of RNA sequences generated in the post-genomic age, many computational methods have been proposed for identifying various types of RNA modifications one by one. However, so far no method whatsoever has been developed for simultaneously identifying several different types of RNA modifications. To address such a challenge, we developed a predictor called “iRNA-3typeA”, by which we can simultaneously identify the occurrence sites of the following three most frequently observed modifications in RNA: (1) N1-methyladenosine (m1A), (2) N6-methyladenosine (m6A), and (3) adenosine to inosine (A-to-I). It has been shown via rigorous cross-validations for the RNA sequences from Homo sapiens and Mus musculus transcriptomes that the success rates achieved by the powerful new predictor are quite high. For the convenience of broad experimental scientists, a user-friendly web-server for iRNA-3typeA has been established at http://lin-group.cn/server/iRNA-3typeA/. It is anticipated that iRNA-3typeA may become a useful high throughput tool for genome analysis.
Targeting Nicotinamide N-Methyltransferase and miR-449a in EGFR-TKI-Resistant Non-Small Cell Lung Cancer Cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-29 Duc-Hiep Bach, Donghwa Kim, Song Yi Bae, Won Kyung Kim, Ji-Young Hong, Hye-Jung Lee, Nirmal Rajasekaran, Soonbum Kwon, Yanhua Fan, Thi-Thu-Trang Luu, Young Kee Shin, Jeeyeon Lee, Sang Kook Lee
Development of a reporter system to explore MMEJ in the context of replacing large genomic fragments Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-22 Mert Yanik, Surya Prakash Goud Ponnam, Tobias Wimmer, Lennart Trimborn, Carina Müller, Isabel Gambert, Johanna Ginsberg, Annabella Janise, Janina Domicke, Wolfgang Wende, Birgit Lorenz, Knut Stieger
Common genome editing strategies are either based on nonhomologous endjoining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology directed repair HDR) or short (microhomology mediated end-joining MMEJ) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ post CRISPR/Cas9 mediated cleavage in cell culture. Following successful prove of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by micro-homologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse RPGR-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line. Luciferase signal appearance represented successful recombination events and was highest when the mhs were 5 bp long, while longer mhs revealed lower luciferase signal. In addition, presence of Csy4 RNase was shown to increase luciferase signaling. The luciferase reporter system is a valuable tool to study the input of the different DNA repair mechanisms in the replacement of large DNA sequences by microhomologous sequences.
Hydrogel-assisted antisense LNA gapmer delivery for in situ gene silencing in spinal cord injury Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-20 Pedro M.D. Moreno, Ana R. Ferreira, Daniela Salvador, Maria T. Rodrigues., Marília Torrado, Eva D. Carvalho, Ulf Tedebark, Mónica M. Sousa, Isabel F. Amaral, Jesper Wengel, Ana P. Pêgo
After spinal cord injury (SCI) nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favourable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration - RhoA and GSK3β. The fibrin matrix assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% down-regulation 5 days after hydrogel injection. Our results show that local implantation of a AON gapmer loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.
CRISPR/Cas9 mediated disruption of the Swedish APP allele as a therapeutic approach for early-onset Alzheimer’s disease Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-16 B. György, C. Lööv, M.P. Zaborowski, S. Takeda, B.P. Kleinstiver, C. Commins, K. Kastanenka, D. Mu, A. Volak, V. Giedraitis, L. Lannfelt, C.A. Maguire, J.K. Joung, B.T. Hyman, X.O. Breakefield, M. Ingelsson
The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using clustered regularly interspaced short palindromic repeats (CRISPR). By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof of concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo - and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.
Novel DNA aptamers that bind to mutant huntingtin and modify its activity Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-16 Baehyun Shin, Roy Jung, Hyejin Oh, Gwen E. Owens, Hyeongseok Lee, Seung Kwak, Ramee Lee, Susan L. Cotman, Jong-Min Lee, Marcy E. MacDonald, Ji-Joon Song, Ravi Vijayvargia, Ihn Sik Seong
The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington’s Disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the carboxyl-terminal CTD-II domain. Aptamer-binding to mutant huntingtin, abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory-activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPC), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPC against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development.
Gelofusine attenuates tubulointerstitial injury induced by cRGD-conjugated siRNA via regulating the TLR3 signaling pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-03-14 Bohong Cen, Wenjie Liao, Zhen Wang, Linyuan Gao, Yuanyi Wei, Wen Huang, Shuai He, Wei Wang, Xiaoxia Liu, Xinghua Pan, Aimin Ji
Integrin αvβ3, which is selectively targeted by cyclic arginine-glycine-aspartic acid (cRGD) peptides, is significantly up-regulated in tumor. Previous studies showed that small interfering RNA (siRNA) modified with cRGD (cRGD–siRNA) could significantly inhibit tumor growth through RNA interference (RNAi) of oncogene expression. However, cRGD–siRNA is partially reabsorbed and trapped in the kidneys, causing renal injury in an unpredictable manner. This study aimed to investigate the influence of Gelofusine on tubulointerstitial injury induced by cRGD–siRNA in vitro and in vivo. Besides, the effect of Gelofusine on the distribution of cRGD–siRNA in tumor-bearing nude mice and wild-type mice was also explored. We found that Gelofusine inhibited the apoptosis and activation of innate immune response of human tubular epithelial cells induced by cRGD–siRNA in vitro. In addition, co-injection of Gelofusine efficiently reduced the renal retention of cRGD–siRNA without affecting its tumor targeting in vivo. Further in vivo studies indicated that Gelofusine significantly attenuated tubulointerstitial injury induced by cRGD–siRNA through regulating Toll-like receptor (TLR)3-mediated activation of nuclear factor-kappa B (NF-κB) and caspase-3 apoptotic pathway. In conclusion, Gelofusine, acting as a novel and effective renal protective agent, could form a compound preparation with siRNA drugs for future clinical applications.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
- Acc. Chem. Res.
- ACS Appl. Mater. Interfaces
- ACS Biomater. Sci. Eng.
- ACS Catal.
- ACS Cent. Sci.
- ACS Chem. Biol.
- ACS Chem. Neurosci.
- ACS Comb. Sci.
- ACS Earth Space Chem.
- ACS Energy Lett.
- ACS Infect. Dis.
- ACS Macro Lett.
- ACS Med. Chem. Lett.
- ACS Nano
- ACS Omega
- ACS Photonics
- ACS Sens.
- ACS Sustainable Chem. Eng.
- ACS Synth. Biol.
- Acta Biomater.
- Acta Crystallogr. A Found. Adv.
- Acta Mater.
- Adv. Colloid Interface Sci.
- Adv. Electron. Mater.
- Adv. Energy Mater.
- Adv. Funct. Mater.
- Adv. Healthcare Mater.
- Adv. Mater.
- Adv. Mater. Interfaces
- Adv. Opt. Mater.
- Adv. Sci.
- Adv. Synth. Catal.
- AlChE J.
- Anal. Bioanal. Chem.
- Anal. Chem.
- Anal. Chim. Acta
- Anal. Methods
- Angew. Chem. Int. Ed.
- Annu. Rev. Anal. Chem.
- Annu. Rev. Biochem.
- Annu. Rev. Environ. Resour.
- Annu. Rev. Food Sci. Technol.
- Annu. Rev. Mater. Res.
- Annu. Rev. Phys. Chem.
- Appl. Catal. A Gen.
- Appl. Catal. B Environ.
- Appl. Clay. Sci.
- Appl. Energy
- Aquat. Toxicol.
- Arab. J. Chem.
- Asian J. Org. Chem.
- Atmos. Environ.
- Carbohydr. Polym.
- Catal. Commun.
- Catal. Rev. Sci. Eng.
- Catal. Sci. Technol.
- Catal. Today
- Cell Chem. Bio.
- Cem. Concr. Res.
- Ceram. Int.
- Chem. Asian J.
- Chem. Bio. Drug Des.
- Chem. Biol. Interact.
- Chem. Commun.
- Chem. Educ. Res. Pract.
- Chem. Eng. J.
- Chem. Eng. Sci.
- Chem. Eur. J.
- Chem. Mater.
- Chem. Phys.
- Chem. Phys. Lett.
- Chem. Phys. Lipids
- Chem. Rev.
- Chem. Sci.
- Chem. Soc. Rev.
- Chin. J. Chem.
- Combust. Flame
- Compos. Part A Appl. Sci. Manuf.
- Compos. Sci. Technol.
- Compr. Rev. Food Sci. Food Saf.
- Comput. Chem. Eng.
- Constr. Build. Mater.
- Coordin. Chem. Rev.
- Corros. Sci.
- Crit. Rev. Food Sci. Nutr.
- Crit. Rev. Solid State Mater. Sci.
- Cryst. Growth Des.
- Curr. Opin. Chem. Eng.
- Curr. Opin. Colloid Interface Sci.
- Curr. Opin. Environ. Sustain
- Curr. Opin. Solid State Mater. Sci.
- Ecotox. Environ. Safe.
- Electrochem. Commun.
- Electrochim. Acta
- Energy Environ. Sci.
- Energy Fuels
- Energy Storage Mater.
- Environ. Impact Assess. Rev.
- Environ. Int.
- Environ. Model. Softw.
- Environ. Pollut.
- Environ. Res.
- Environ. Sci. Policy
- Environ. Sci. Technol.
- Environ. Sci. Technol. Lett.
- Environ. Sci.: Nano
- Environ. Sci.: Processes Impacts
- Environ. Sci.: Water Res. Technol.
- Eur. J. Inorg. Chem.
- Eur. J. Med. Chem.
- Eur. J. Org. Chem.
- Eur. Polym. J.
- J. Acad. Nutr. Diet.
- J. Agric. Food Chem.
- J. Alloys Compd.
- J. Am. Ceram. Soc.
- J. Am. Chem. Soc.
- J. Am. Soc. Mass Spectrom.
- J. Anal. Appl. Pyrol.
- J. Anal. At. Spectrom.
- J. Antibiot.
- J. Catal.
- J. Chem. Educ.
- J. Chem. Eng. Data
- J. Chem. Inf. Model.
- J. Chem. Phys.
- J. Chem. Theory Comput.
- J. Chromatogr. A
- J. Chromatogr. B
- J. Clean. Prod.
- J. CO2 UTIL.
- J. Colloid Interface Sci.
- J. Comput. Chem.
- J. Cryst. Growth
- J. Dairy Sci.
- J. Electroanal. Chem.
- J. Electrochem. Soc.
- J. Environ. Manage.
- J. Eur. Ceram. Soc.
- J. Fluorine Chem.
- J. Food Drug Anal.
- J. Food Eng.
- J. Food Sci.
- J. Funct. Foods
- J. Hazard. Mater.
- J. Heterocycl. Chem.
- J. Hydrol.
- J. Ind. Eng. Chem.
- J. Inorg. Biochem.
- J. Magn. Magn. Mater.
- J. Mater. Chem. A
- J. Mater. Chem. B
- J. Mater. Chem. C
- J. Mater. Process. Tech.
- J. Mech. Behav. Biomed. Mater.
- J. Med. Chem.
- J. Membr. Sci.
- J. Mol. Catal. A Chem.
- J. Mol. Liq.
- J. Nat. Gas Sci. Eng.
- J. Nat. Prod.
- J. Nucl. Mater.
- J. Org. Chem.
- J. Photochem. Photobiol. C Photochem. Rev.
- J. Phys. Chem. A
- J. Phys. Chem. B
- J. Phys. Chem. C
- J. Phys. Chem. Lett.
- J. Polym. Sci. A Polym. Chem.
- J. Porphyr. Phthalocyanines
- J. Power Sources
- J. Solid State Chem.
- J. Taiwan Inst. Chem. E.
- Macromol. Rapid Commun.
- Mass Spectrom. Rev.
- Mater. Chem. Front.
- Mater. Des.
- Mater. Horiz.
- Mater. Lett.
- Mater. Sci. Eng. A
- Mater. Sci. Eng. R Rep.
- Mater. Today
- Meat Sci.
- Med. Chem. Commun.
- Microchem. J.
- Microchim. Acta
- Micropor. Mesopor. Mater.
- Mol. Biosyst.
- Mol. Cancer Ther.
- Mol. Catal.
- Mol. Nutr. Food Res.
- Mol. Pharmaceutics
- Mol. Syst. Des. Eng.
- Nano Energy
- Nano Lett.
- Nano Res.
- Nano Today
- Nano-Micro Lett.
- Nanomed. Nanotech. Biol. Med.
- Nanoscale Horiz.
- Nat. Catal.
- Nat. Chem.
- Nat. Chem. Biol.
- Nat. Commun.
- Nat. Energy
- Nat. Mater.
- Nat. Med.
- Nat. Methods
- Nat. Nanotech.
- Nat. Photon.
- Nat. Prod. Rep.
- Nat. Protoc.
- Nat. Rev. Chem.
- Nat. Rev. Drug. Disc.
- Nat. Rev. Mater.
- Natl. Sci. Rev.
- Neurochem. Int.
- New J. Chem.
- NPG Asia Mater.
- npj 2D Mater. Appl.
- npj Comput. Mater.
- npj Flex. Electron.
- npj Mater. Degrad.
- npj Sci. Food
- Pharmacol. Rev.
- Pharmacol. Therapeut.
- Photochem. Photobiol. Sci.
- Phys. Chem. Chem. Phys.
- Phys. Life Rev.
- PLOS ONE
- Polym. Chem.
- Polym. Degrad. Stabil.
- Polym. J.
- Polym. Rev.
- Powder Technol.
- Proc. Combust. Inst.
- Prog. Cryst. Growth Ch. Mater.
- Prog. Energy Combust. Sci.
- Prog. Mater. Sci.
- Prog. Photovoltaics
- Prog. Polym. Sci.
- Prog. Solid State Chem.
- Sci. Adv.
- Sci. Bull.
- Sci. Rep.
- Sci. Total Environ.
- Sci. Transl. Med.
- Scr. Mater.
- Sens Actuators B Chem.
- Sep. Purif. Technol.
- Small Methods
- Soft Matter
- Sol. Energy
- Sol. Energy Mater. Sol. Cells
- Solar RRL
- Spectrochim. Acta. A Mol. Biomol. Spectrosc.
- Surf. Sci. Rep.
- Sustainable Energy Fuels