RNA replacement therapy utilizing transcript knockdown and an optimized resistant transgene for rescue of oculopharyngeal muscular dystrophy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-15 Aida Abu-Baker, Nawwaf Kharma, Jonathan Perreault, Alanna Grant, Masoud Shekarabi, Claudia Maios, Michele Dona, Christian Neri, Patrick A. Dion, Alex Parker, Luc Varin, Guy A. Rouleau
MicroRNA-126 regulates angiogenesis and neurogenesis in a mouse model of focal cerebral ischemia Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-11 Meijie Qu, Jiaji Pan, Liping Wang, Panting Zhou, Yaying Song, Shuhong Wang, Lu Jiang, Jieli Geng, Zhijun Zhang, Yongting Wang, Yaohui Tang, Guo-Yuan Yang
Studies demonstrate that microRNA-126 plays a critical role in promoting angiogenesis. However, its effects on angiogenesis following ischemic stroke is unclear. Here, we explored the effect of microRNA-126-3p and -5p on angiogenesis and neurogenesis after brain ischemia. We demonstrated that both miRNA-126-3p and -5p increased the proliferation, migration, and tube formation of HUVECs compared to the scrambled miRNA control (p<0.05). Transferring microRNA-126 into a mouse middle cerebral artery occlusion model via lentivirus, we found that microRNA-126 overexpression increased the number of CD31+/BrdU+ proliferating endothelial cells and DCX+/BrdU+ neuroblasts in the ischemic mouse brain, improved neurobehavioral outcomes (p<0.05), and reduced brain atrophy volume (p<0.05) compared to control mice. Western blot results showed that AKT and ERK signaling pathways were activated in lentiviral-microRNA-126 treated group (p<0.05). Both PCR and western blot results demonstrated that tyrosine-protein phosphatase non-receptor type 9 (PTPN9) was decreased in lentiviral-microRNA-126 treated group (p<0.05). Dual-luciferase gene reporter assay also showed that PTPN9 was the direct target of microRNA-126-3p and -5p in the ischemic brain. We demonstrated that microRNA-126-3p and -5p promoted angiogenesis and neurogenesis in ischemic mouse brain, and further improved neurobehavioral outcomes. Our mechanistic study further showed that microRNA-126 mediated angiogenesis through directly inhibiting its target PTPN9 and activating AKT and ERK signaling pathways.
Targeting RNA-mediated toxicity in C9ORF72 ALS/FTD by RNAi-based gene therapy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-11 Raygene Martier, Jolanda M. Liefhebber, Ana García-Osta, Jana Miniarikova, Mar Cuadrado-Tejedor, Maria Espelosin, Susana Ursua, Harald Petry, Sander J. van Deventer, Melvin M. Evers, Pavlina Konstantinova
A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9ORF72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial miRNAs (miC) targeting C9ORF72 to reduce gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of AAV5-miC in human-derived iPSC-neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9ORF72 transcripts. Additionally, we demonstrated silencing of C9ORF72 in both nucleus and cytoplasm which has an added value for treatment of ALS/FTD patients. A proof of concept in an ALS mouse model demonstrated significant reduction of the repeat-containing C9ORF72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on reduction of toxicity caused by the repeat-containing C9ORF72 transcripts.
Discovery of the oncogenic parp1, a target of bcr-abl and a potential therapeutics, in identification of mir-181a/PPFIA1 signaling pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-08 Chunming Gu, Yanjun Liu, Zhao Yin, Juhua Yang, Guiping Huang, Xuejiao Zhu, Yumin Li, Jia Fei
miR-181a is downregulated in leukemia and affects their progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that PPFIA1 is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of NF-κB-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.
Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-07 Kimberly J. Hassett, Kerry E. Benenato, Eric Jacquinet, Aisha Lee, Angela Woods, Olga Yuzhakov, Sunny Himansu, Jessica Deterling, Benjamin M. Geilich, Tatiana Ketova, Cosmin Mihai, Andy Lynn, Iain McFadyen, Melissa Moore, Joe Senn, Matthew G. Stanton, Örn Almarsson, Giuseppe Ciaramella, Luis A. Brito
MEG3 as a competing endogenous RNA binds with miR-27a to promote PHLPP2 protein translation and impairs bladder cancer invasion Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-07 Chao Huang, Xin Liao, Honglei Jin, Fei Xie, Fuxing Zheng, Jingxia Li, Chenfan Zhou, Guosong Jiang, Xue-Ru Wu, Chuanshu Huang
Muscle invasive and metastatic bladder cancer have an extremely poor 5-year survival rate 5%. In comparison, there is a 77% 5-year survival rate from all other bladder cancers (BCs). This striking contrast indicates that one of the therapeutic kernels for BC is to elucidate the molecular mechanisms underlying invasiveness and metastasis of BC. In the current study, we demonstrate that maternally expressed gene 3 (MEG3) is significantly downregulated in human invasive BCs in comparison to non-invasive BCs, and that ectopic expression of MEG3 dramatically inhibits the invasiveness of human BC cells. Consistently, ectopic expression of MEG3 also attenuates metastatic ability of T24T cells, a cell line derived from T24 cells, in the lungs of nude mice. Our mechanistic studies reveal that MEG3, as a ceRNA, inhibits the invasiveness of human BC cells via negative regulation of c-Myc by competing with PHLPP2 mRNA for miR-27a. These findings not only provide a novel insight into understanding the mechanisms behind the MEG3 inhibition of BC cell invasion, it also reveals the potential use of MEG3 as a potential tool for the prevention and therapy of invasive BC.
Suppression of choroidal neovascularization by AAV-based dual-acting antiangiogenic gene therapy Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-02 Anne Louise Askou, Sidsel Alsing, Josephine N.E. Benckendorff, Andreas Holmgaard, Jacob Giehm Mikkelsen, Lars Aagaard, Toke Bek, Thomas J. Corydon
Vascular endothelial growth factor A (VEGFA) is involved in the pathogenesis of vasoproliferative retinal diseases such as exudative age-related macular degeneration (AMD). The objective of this study was to investigate whether dual-acting therapy based on simultaneous expression of anti-VEGFA microRNAs (miRNA) and the secreted, antiangiogenic protein pigment endothelial-derived factor (PEDF) delivered by adeno-associated virus (AAV) vectors provides improved protection against choroidal neovascularization (CNV). To investigate this, a multigenic AAV vector allowing retina pigment epithelium (RPE)-specific expression of anti-VEGFA miRNAs and PEDF was engineered. Robust expression of PEDF, driven by the RPE-specific vitelliform macular dystrophy 2 promoter, was observed in human cells and in mouse retina. Significant reduction of CNV was observed in a laser-induced CNV mouse model 57 days post injection of AAV5-particles conveying either anti-VEGFA miRNA and PEDF dual-therapy or anti-VEGFA miRNA mono-therapy. Overall, CNV reduction was most prominent in animals receiving dual-acting therapy. In both cases reduction of CNV was accompanied by a significant attenuation of VEGFA. In conclusion, the presented data reveals that gene therapy targeting VEGFA via multigenic AAV vectors displays combined efficacy, suggesting that dual-acting therapy is an important tool in future eye gene therapy for treatment of neovascular ocular diseases, including AMD.
Circulating long non-coding RNA lncRNA-BC030099 increases in preeclampsia patients Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-02-01 Yuhong Sun, Yan Hou, Nan lv, Qian Liu, Nan lin, Shuyu Zhao, Xiaodan Chu, Xuan Chen, Guobin Cheng, Peiling Li
Long noncoding RNAs (lncRNAs) have increasingly been shown to be important biological regulators involved in numerous diseases. Further, increasing evidence demonstrates that circulating lncRNAs can be used as diagnostic biomarkers. Therefore, the purpose of this study was to evaluate the potential for circulating lncRNAs as novel biomarkers for the diagnosis of preeclampsia. In the present study, we measured the expression of five lncRNAs known to be relevant to the uterus in whole blood samples from 48 preeclampsia patients and 24 non-preeclampsia healthy subjects using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). We found that circulating levels of lncRNA-BC030099 were significantly higher in patients with preeclampsia (1.232 ± 0.4870) than in non-preeclampsia healthy subjects (0.9928 ± 0.2008, P < 0.05). The area under the receiver operating characteristic (ROC) curve for lncRNA-BC030099 was 0.713. Univariate and multivariate analyses identified lncRNA-BC030099 as an independent predictor for preeclampsia. In brief, our results suggest that increased plasma levels of lncRNA-BC030099 are associated with an increased risk of preeclampsia and may be considered a novel biomarker.
Artificial microRNAs targeting C9ORF72 have the potential to reduce accumulation of the intra-nuclear transcripts in ALS and FTD patients Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-30 Raygene Martier, Jolanda M. Liefhebber, Jana Miniarikova, Tom van der Zon, Jolanda Snapper, Iris Kolder, Harald Petry, Sander J. van Deventer, Melvin M. Evers, Pavlina Konstantinova
The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA-seq data of C9ORF72-ALS patients and controls were analyzed to better understand the sequence conservation of C9ORF72 in patients. MicroRNAs were developed in conserved regions to silence C9ORF72 (miC) and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by reduction of endogenously C9ORF72 mRNA in both nucleus and cytoplasm and ∼50% reduction of nuclear RNA foci in (G4C2)44 expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated viral vector serotype 5 (AAV5) and silencing of C9ORF72 was demonstrated in HEK293T cells and Induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of miRNA-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.
The miR-223 regulates cell proliferation and invasion via targeting PDS5B in pancreatic cancer cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-29 Jia Ma, Tong Cao, Yue Cui, Fan Zhang, Ying Shi, Jun Xia, Z Peter Wang
Emerging evidence has demonstrated that miR-223 is critically involved in the progression of pancreatic cancer (PC); however, the underlying mechanisms are not fully elucidated. In the current study, we explored the molecular basis of miR-223-mediated tumor progression in PC. We performed numerous approaches including MTT, FACS, Transfection, RT-PCR, Western blotting, Transwell, and animal studies to determine the physiological role of miR-223 in PC cells. We found that sister chromatid cohesion protein PDS5 homolog B (PDS5B) is a direct target of miR-223 in PC. Moreover, PDS5B exhibits tumor suppressive function in PC cells. Consistently, ectopic overexpression of PDS5B reversed miR-223-mediated tumor progression in PC cells. These results suggest that the miR-223/PDS5B axis regulates cell proliferation and invasion in pancreatic cancer cells. Our findings indicated that down-regulation of miR-223 could be a novel therapeutic approach for PC.
Scavenger receptor class A1 mediates uptake of morpholino antisense oligonucleotide into dystrophic skeletal muscle Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-25 Shouta Miyatake, Yoshitaka Mizobe, Maria Tsoumpra, Kenji Rowel Q. Lim, Yuko Hara, Fazel Shabanpoor, Toshifumi Yokota, Shin’ichi Takeda, Yoshitsugu Aoki
Exon-skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD.
Bioengineered Let-7c Inhibits Orthotopic Hepatocellular Carcinoma and Improves Overall Survival with Minimal Immunogenicity Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-24 Joseph L. Jilek, Qian-Yu Zhang, Mei-Juan Tu, Pui Yan Ho, Zhijian Duan, Jing-Xin Qiu, Ai-Ming Yu
Exosomal Transmission of MicroRNA from HCV Replicating Cells Stimulates Transdifferentiation in Hepatic Stellate Cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-18 Ji Hyun Kim, Chang Ho Lee, Seong-Wook Lee
TFAP2C-activated MALAT1 modulates the chemoresistance of docetaxel-resistant lung adenocarcinoma cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-18 Jing Chen, Xiaobei Liu, Yichen Xu, Kai Zhang, Jiayuan Huang, Banzhou Pan, Dongqin Chen, Shiyun Cui, Haizhu Song, Rui Wang, Xiaoyuan Chu, Xiaoli Zhu, Longbang Chen
Chemoresistance remains a great obstacle in effective lung adenocarcinoma (LUAD) treatment. Previously, we verified the role of microRNA-200b (miR-200b) in the formation of docetaxel (DTX)-resistant LUAD cells. This study aims to investigate the mechanism underlying the low level of miR-200b in DTX-resistant LUAD cells. RT2 lncRNA PCR array system was applied to explore lncRNAs which potentially regulated miR-200b in DTX-resistant LUAD cells. MALAT1 contributed to the low miR-200b level in DTX-resistant LUAD cells. Functional assays were conducted to determine the role of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in regulating the growth and metastasis of parental and DTX-resistant LUAD cells. Mechanism investigation revealed the mechanism of competing endogenous RNA (ceRNA) pathway. MALAT1 regulated miR-200b by acting as a ceRNA. MALAT1 modulated the sensitivity of LUAD cells to DTX. E2F transcription factor 3 (E2F3) and zinc finger E-box binding homeobox 1 (ZEB1) were two targets of miR-200b and mediated the function of MALAT1 in DTX-resistant LUAD cells. Transcription factor AP-2 gamma (TFAP2C) and ZEB1 activated the MALAT1 transcription. In conclusion, TFAP2C-activated MALAT1 modulated the chemoresistance of LUAD cells by sponging miR-200b to upregulate E2F3 and ZEB1. Our findings may provide novel therapeutic targets and perspectives for LUAD treatment.
CircRNA RAPGEF5 contributes to papillary thyroid cancer cell proliferation, invasion, and migration by sponging miR-198 and promoting FGFR1 expression Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-15 Weiwei Liu, Ji Zhao, Mingming Jin, Ming Zhou
The circular RNA RAPGEF5 (circRAPGEF5) is generated from five exons of the RAPGEF5 gene and abnormal expression in papillary thyroid cancer (PTC). However, if circRAPGEF5 play a role in PTC tumorigenesis remains unclear. The aim of the present study was to investigate the role of circRAPGEF5 in PTC. The results showed that circRAPGEF5 was upregulated in PTC tissues and cell lines. CircRAPGEF5 knockdown inhibited cell proliferation, migration, and invasion in vitro. CircRAPGEF5 silencing downregulated fibroblast growth factor receptor 1 (FGFR1) expression by ‘sponging’ miR-198, suppressing the aggressive biological behaviors of PTC. Luciferase reporter assays confirmed that circRAPGEF5 interacted with miR-198, and miR-198 interacted with the 3'-UTR of FGFR1 to downregulate its expression. Xenograft experiments confirmed that circRAPGEF5 knockdown suppressed FGFR1 mediated tumor growth by promoting miR-198 expression. CircRAPGEF5 acts as a tumor promoter via a novel circRAPGEF5/miR-198/FGFR1 axis, providing a potential biomarker and therapeutic target for the management of PTC.
miR-582-3p and -5p Suppress Prostate Cancer Metastasis to Bone by repressing TGF-β signaling Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-15 Shuai Huang, Changye Zou, Yubo Tang, Qingde Wa, Xinsheng Peng, Xiao Chen, Chunxiao Yang, Dong Ren, Yan Huang, Zhuangwen Liao, Sheng Huang, Xuenong Zou, Jincheng Pan
A number of evidence has reported that aberrant expression of miRNAs closely correlates with the bone metastasis of prostate cancer (PCa.). However, clinical significance and functional roles of both strands of a single miRNA in bone metastasis of PCa remain undefined. Here, we reported that miR-582-3p and -5p expression were simultaneously reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Downexpression of miR-582-3p and -5p strongly and positively correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-582-3p and -5p inhibited invasion and migration abilities of PCa cells in vitro, as well as repressed bone metastasis in vivo. Our results further revealed that miR-582-3p and -5p attenuated bone metastasis of PCa via inhibiting TGF-β signaling by simultaneously targeting several components of TGF-β signaling, including SMAD2, SMAD4, TGF-βreceptor I (TGFBRI) and TGFBRII. Moreover, deletion contributes to miR-582-3p and -5p downexpression in PCa tissues. Finally, clinical negative correlations of miR-582-3p and -5p with SMAD2, SMAD4, TGFBRI and TGFBRII were demonstrated in PCa tissues. Thus, our findings explore a novel tumor suppressive miRNA with its both strands implicated in bone metastasis of PCa, suggesting its potential therapeutic value in treatment of PCa bone metastasis.
Knockdown of USF1 inhibits the vasculogenic mimicry of glioma cells via stimulating SNHG16/miR-212-3p and linc00667/miR-429 axis Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-15 Di Wang, Jian Zheng, Xiaobai Liu, Yixue Xue, Libo Liu, Jun Ma, Qianru He, Zhen Li, Heng Cai, Yunhui Liu
The anti-angiogenic treatment of malignant glioma cells is an effective method to treat high-grade gliomas. However, due to the presence of vasculogenic mimicry (VM), the anti-angiogenic treatment of gliomas is not significantly effective in improving overall patient median survival. Therefore, this study investigated the mechanism of mimic formation of angiogenesis in gliomas. The results of this experiment indicate that the expression of USF1 is up-regulated in glioma tissues and cells. USF1 knockdown inhibits the proliferation, migration, invasion, VM, and expression of VM-associated proteins in glioma cells by stimulating SNHG16 and linc00667. These two lncRNAs regulate ALHD1A1 through the ceRNA mechanism influencing the VM of glioma. This study is the first to demonstrate that the USF1/SNHG16/miR-212-3p/ALDH1A1 and USF1/linc00667/miR-429/ ALDH1A1 axis regulates the VM of glioma cells, and these findings might provide a novel strategy for glioma treatment.
Simultaneous suppression of multiple programmed cell death pathways by microRNA-105 in cardiac ischemic injury Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-10 Sunhye Shin, Jung-Won Choi, Hanbyeol Moon, Chang Youn Lee, Jun-Hee Park, Jiyun Lee, Hyang-Hee Seo, Gyoonhee Han, Soyeon Lim, Seahyoung Lee, Sang Woo Kim, Ki-Chul Hwang
Recent studies have shown that several upstream signaling elements of apoptosis and necroptosis are closely associated with acute injury in the heart. In our study, we observed that miR-105 was notably dysregulated in rat hearts with myocardial infarction (MI). Thus, the purpose of this study was to test the hypothesis that miR-105 participates in the regulation of RIP3/p-MLKL- and BNIP3-dependent necroptosis/apoptosis in H9c2 cells and MI rat hearts. Our results show that the RIP3/p-MLKL necroptotic pathway and BNIP3-dependent apoptosis signaling are enhanced in H9c2 cells under hypoxic conditions, whereas compared with these pathways in the controls, those in miR-105-treated H9c2 cells are suppressed. Mechanistically, we identified miR-105 as the miRNA directly suppressing the expression of RIP3 and BNIP3, two important mediators involved in cell necroptosis and apoptosis. Furthermore, MI rat hearts injected with miR-105 had decreased infarct sizes, indicating that miR-105 is among three miRNAs that function simultaneously to suppress necroptotic/apoptotic cell death pathways and to inhibit MI-induced cardiomyocyte cell death at multiple levels. Taken together, miR-105 may constitute a new therapeutic strategy for cardioprotection in ischemic heart disease.
Downregulation of MicroRNA hsa-miR-340-5p in Influenza A Virus Infected A549 Cells Suppresses Viral Replication by Targeting RIG-I and OAS2 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-10 Lianzhong Zhao, Xiaohan Zhang, Zhu Wu, Kun Huang, Xiaomei Sun, Huanchun Chen, Meilin Jin
The influenza A virus poses serious public health challenges worldwide. Strikingly, small noncoding microRNAs (miRNAs) that modulate gene expression are closely involved in antiviral responses, although the underlying mechanisms are essentially unknown. We now report that microRNA-340 (miR340) is downregulated following influenza A and other RNA virus infections, implying that host cells deplete miR340 as an antiviral defense mechanism. Accordingly, the inhibition or knockdown of endogenous miR340 clearly prevents the infection of cultured cells, whereas the forced expression of miR340 significantly enhances virus replication. Using next-generation sequencing, we found that miR340 attenuates cellular antiviral immunity. Moreover, mechanistic studies defined miR340 as a repressor of RIG-I and OAS2, critical factors for the establishment of an antiviral response. Collectively, these data indicate that host cells may lower their viral loads by regulating miRNA pathways, which may, in turn, provide new opportunities for treatment.
ERK Inhibitor Enhances Everolimus Efficacy through Attenuation of dNTP Pools in Renal Cell Carcinoma Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-10 Yun Zou, Wenzhi Li, Juan Zhou, Jin Zhang, Yiran Huang, Zhong Wang
The clinical efficiency of everolimus, an mTOR inhibitor, is palliative as sequential or second-line therapy for renal cell carcinoma (RCC). However, the limited response of everolimus in RCC remains uncertain. In the present study, everolimus-resistant RCC models were established to understand the mechanisms and to seek combination approaches. Consequently, activation of ERK was found to contributes towards everolimus-acquired resistance and poor prognosis in patients with RCC. In addition, the efficacy and mechanism of combination treatment underlying RCC using everolimus and ERK inhibitors was investigated. The ERK inhibitor in combination with everolimus synergistically inhibited the proliferation of RCC cells by arresting the cell cycle in the G1 phase. The combination treatment markedly attenuated the dNTP pools by downregulating the mRNA expression of RRM1 and RRM2 through E2F1. The overexpression of E2F1 or supplementation of dNTP rescued the anti-proliferation activity of the everolimus-SCH772984 combination. The antitumor efficacy of combination therapy was reiterated in RCC xenograft models. Thus, the current findings provided evidence that the everolimus-ERK inhibitor combination is a preclinical therapeutic strategy for RCC.
MicroRNA-672-5p identified during weaning reverses osteopenia and sarcopenia in ovariectomized mice Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-10 Naseer Ahmad, Priyanka Kushwaha, Anirudha Karvande, Ashish Kumar Tripathi, Priyanka Kothari, Sulekha Adhikary, Vikram Khedgikar, Vijay Kumar Mishra, Ritu Trivedi
Human novel microRNA seq-915_x4024 in keratinocytes contributes to skin regeneration by suppressing the inflammatory response and TGF-β1/Smad signaling pathway-induced scar formation Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2019-01-10 Feng Zhao, Hongxin Lang, Zhe Wang, Tao Zhang, Dianbao Zhang, Rui Wang, Xuewen Lin, Xiaoyu Liu, Ping Shi, Xining Pang
Early in gestation, wounds in fetal skin heal by regeneration, in which microRNAs play key roles. Seq-915_x4024 is a novel microRNA candidate confirmed by deep sequencing and mirTools 2.0. It is highly expressed in fetal keratinocytes during early gestation. Using an in vitro wound healing assay, transwell cell migration assay and MTS proliferation assay, we demonstrated that keratinocytes overexpressing seq-915_x4024 exhibited higher proliferative activity and the ability to promote fibroblast migration and fibroblast proliferation. These characteristics of keratinocytes are the same biological behaviors as those of fetal keratinocytes, which contribute to skin regeneration. In addition, seq-915_x4024 suppressed the expression of the pro-inflammatory markers TNF-α, IL-6 and IL-8 and the pro-inflammatory chemokines CXCL1 and CXCL5. We also demonstrated that seq-915_x4024 regulates TGF-β isoforms and the extracellular matrix. Moreover, using an in vivo wound healing model, we demonstrated that overexpression of seq-915_x4024 in keratinocytes suppresses inflammatory cell infiltration and scar formation. Using bioinformatics analyses, luciferase reporter assays and Western blotting, we further demonstrated that Sar1A, Smad2, TNF-α and IL-8 are direct targets of seq-915_x4024. Furthermore, the expression of phosphorylated Smad2 and Smad3 was reduced by seq-915_x4024. Seq-915_x4024 could be used as an anti-fibrotic factor for the treatment of wound healing.
Hypermethylation of miR-205-5p by IR governs aggressiveness and metastasis via regulating Bcl-w and Src Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-31 Eun Sook Kim, Jae Yeon Choi, Su Jin Hwang, In Hwa Bae
Although radiotherapy has been successfully applied to treat many cancer types, surviving cancer cells often acquire therapeutic resistance, leading to increased risk of local recurrence and distant metastases via modification of the tumor microenvironment. Earlier, we reported that high expression of Bcl-w in cancer patients is significantly correlated with poor survival as well as malignant activity. However, the relationship between ionizing radiation (IR)-induced resistance and Bcl-w expression in cancer cells is currently unclear. We showed that IR-induced Bcl-w contributes to EMT (epithelial-mesenchymal transition), migration, angiogenesis, stemness maintenance and metastasis by promoting the expression of factors related to these phenotypes, both in vitro and in vivo. Meanwhile, IR enhanced hypermethylation of miR-205-5p CpG islands through Src activation, leading to decreased miR-205-5p expression and, in turn, potentially stimulating Bcl-w-mediated malignant activity and metastasis. The clinical applicability of Bcl-w and miR-205-5p from cells or animal models was confirmed using tissues and plasma of breast carcinoma patients. Based on the collective findings, we propose that miR-205-5p as important negative mediators of resistance in radiotherapy could serve as useful potential targets of concurrently applied genetic therapy aimed to inhibit tumor aggressiveness and enhance the efficiency of radiotherapy in cancer patients.
miR-10a-5p promotes chondrocyte apoptosis in osteoarthritis by targeting HOXA1 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-25 Yan Ma, Yizheng Wu, Junxin Chen, Kangmao Huang, Bin Ji, Zhijun Chen, Qiang Wang, Jianjun Ma, Shuying Shen, Jianfeng Zhang
Osteoarthritis is a common joint disease characterized by degradation of the articular cartilage and joint inflammation. Studies have revealed the importance of microRNAs in the regulation of chondrocyte apoptosis. MicroRNA deep sequencing of control and osteoarthritic cartilage has revealed that miR-10a-5p is significantly upregulated in osteoarthritic tissues. However, its role in these tissues remains unknown. The present study was conducted to investigate the effect of miR-10a-5p in promoting osteoarthritis. miR-10a-5p expression was increased in chondrocytes after interleukin-1β treatment in vitro. Transfection with an miR-10a-5p inhibitor abrogated interleukin-1β-induced apoptosis. Luciferase activity assay showed that miR-10a-5p targeted the 3′-untranslated region of the homeobox gene HOXA1, inhibiting its expression. Treatment with HOXA1 siRNA reversed the rescuing effect of the miR-10a-5p inhibitor on chondrocyte apoptosis. Additionally, an osteoarthritis model was established in mice by anterior cruciate ligament transection. AntagomiR-10a-5p improved the cartilage surfaces of osteoarthritic mice, whereas agomiR-10a-5p worsened them. A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay indicated reduced apoptosis and increased HOXA1 expression in osteoarthritic mice after miR-10a-5p knockdown. These findings reveal a novel mechanism regulating osteoarthritis progression and demonstrate the potential of miR-10a-5p and homeobox protein HOXA1 as therapeutic targets.
The Attenuation of Trophoblast Invasion Caused by the Downregulation of EZH2 is Involved in the Pathogenesis of Human Recurrent Miscarriage Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-22 Shijian Lv, Na Wang, Hong Lv, Jieqiong Yang, Jianwei Liu, Wei-Ping Li, Cong Zhang, Zi-Jiang Chen
Recurrent miscarriage (RM) is currently defined as two or more losses of a clinically established intrauterine pregnancy. Despite years of research, RM continues to be a clinically frustrating challenge for patients and physicians, and its etiology remains poorly understood. Accumulating evidence has suggested that epigenetic modifications are involved in early embryogenesis, and defects in epigenetic patterning contribute to the development of RM. Here, we studied the role of enhancer of zeste homolog 2 (EZH2) in the pathogenesis of RM and found that the EZH2 expression was significantly decreased in the villi from women with RM compared to that in control villi. EZH2 promoted the invasion of trophoblast cells. Moreover, EZH2 could promote epithelial-mesenchymal transition by epigenetically silencing CDX1. Both ChIP-PCR and dual-luciferase report assays demonstrated that EZH2 repressed CDX1 transcription via direct binding to its promoter region and then trimethylating Histone3-Lysine27. Furthermore, we discovered that progesterone which is used extensively in the treatment of miscarriage and RM increased the expression of EZH2 via extracellular signaling-regulated kinase (ERK1/2) pathway. These findings revealed that EZH2 may regulate trophoblast invasion as an epigenetic factor, suggesting that EZH2 might be a potential therapeutic target for RM.
AntagomiR-103/107 treatment affects cardiac function and metabolism Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-22 Monika Rech, Annika R. Kuhn, Joost Lumens, Paolo Carai, Rick van Leeuwen, Wouter Verhesen, Robin Verjans, Julie Lecomte, Yilin Liu, Joost J.F.P. Luiken, Ronny Mohren, Berta Cillero-Pastor, Stephane Heymans, Kèvin Knoops, Marc van Bilsen, Blanche Schroen
MicroRNA-103/107 regulate systemic glucose metabolism and insulin sensitivity. For this reason, inhibitory strategies for these microRNAs are currently being tested in clinical trials. Given the high metabolic demands of the heart and the abundant cardiac expression of miR-103/107, we questioned whether antagomir-mediated inhibition of miR-103/107 in C57Bl/6J mice impacts on cardiac function. Notably, fractional shortening decreased after 6 weeks of antagomiR-103/107 treatment. This was paralleled by a prolonged systolic radial and circumferential time to peak and by a decreased global strain rate. Histology and electron microscopy showed reduced cardiomyocyte area, decreased mitochondrial volume and mitochondrial cristae density following antagomiR-103/107. In line, antagomiR-103/107 treatment decreased mitochondrial OXPHOS complexes protein levels compared to scrambled, as assessed by mass spectrometry-based label-free quantitative proteomics. MiR-103/107 inhibition in primary cardiomyocytes did not affect glycolysis rates, but decreased mitochondrial reserve capacity, reduced mitochondrial membrane potential and altered mitochondrial network morphology as assessed by live cell imaging. Our data indicate that antagomiR-103/107 decrease cardiac function, cardiomyocyte size and mitochondrial oxidative capacity in absence of pathological stimuli. These data raise concern about the possible cardiac implications of the systemic use of antagomiR-103/107 in the clinical setting, and careful cardiac phenotyping within ongoing trials is highly recommended.
Generation of Genetically Stable Human Direct Conversion derived Neural Stem Cells using Quantity Control of Proto-oncogene Overexpression Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-20 Daekee Kwon, Mi-Jung Han, Min-Jun Jee, Hee-Jin Ahn, Kwang-Won Seo, Kyung-Sun Kang
As the human lifespan has increased due to developments in medical technology, the number of patients with neurological diseases has rapidly increased. Therefore, studies on effective treatments for neurological diseases are becoming increasingly important. To perform these studies, it is essential to obtain a large number of patient-derived neural cells. The purpose of the present study was to establish a technology that allows the high-efficiency generation of genetically stable direct conversion derived neural stem cells (dcNSCs) through expression of a new combination of reprogramming factors, including a proto-oncogene. Specifically, human c-Myc proto-oncogene and the human Sox2 gene were overexpressed in a precisely controlled manner in various human somatic cells. As a result, the direct conversion into multipotent dcNSCs occurred only when the cells were treated with a multiplicity of infection (MOI) of 1 of hc-Myc proto-oncogene and hSox2 retrovirus. When MOIs of 5 or 10 were utilized, distinct results were obtained. In addition, the pluripotency was bypassed during this process. Notably, as the MOI used to treat the cells increased, expression of the p53 tumor suppressor gene, which is typically a reprogramming hurdle, increased proportionately. Interestingly, p53 was genetically stable in dcNSCs generated through direct conversion into a low p53 expression state. In the present study, generation of genetically stable dcNSCs using direct conversion was optimized by precisely controlling the overexpression of a proto-oncogene. This method could be utilized in future studies, such as in vitro drug screening using generated dcNSCs. In addition, this method could be effectively utilized in studies on direct conversion into other types of target cells.
Viral vector-based delivery of CRISPR/Cas9 and donor DNA for homology directed repair in an in vitro model for canine hemophilia B Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-20 Jian Gao, Thorsten Bergmann, Wenli Zhang, Maren Schiwon, Eric Ehrke-Schulz, Anja Ehrhardt
Gene therapy represents an attractive alternative to treat hemophilia B. Here we established three hepatocyte-derived cell lines based on Huh7, PLC/PRF/5 and Hep3B cells stably carrying a mutated canine FIX (cFIXmut) transgene containing a single point mutation in the catalytic domain. Based on these in vitro models resembling a commonly used canine large animal model, the Tet-on inducible CRISPR/Cas9 system and an optimized donor were used to correct mutated cFIX gene through homology directed repair (HDR). For efficient delivery of designer nuclease and donor DNA we produced a high-capacity adenovirus vector type 5 (HCAdV5) containing the Tet-on inducible cFIX-specific CRISPR/Cas9 system and a single-stranded adeno-associated virus type 2 vector (ssAAV2) containing the modified donor. Moreover we designed a single HCAdV5 delivering all components for HDR. Our allele refractory mutation detection system based on quantitative PCR analysis (ARMS-qPCR) revealed that the single vector application in Huh7-cFIXmut cells resulted in up to 5.52% HDR efficiencies, which was superior to the two-vector strategy. Furthermore the single vector also resulted in increased phenotypic correction efficiencies assayed by enzyme-linked immunosorbent assay (ELISA). We conclude that HDR in combination with viral vector delivery holds great promise for correction of mutated FIX in disease models.
Nucleotide modification alters microRNA-dependent silencing of microRNA switches Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-18 John Lockhart, John Canfield, Ezinne Francess Mong, Jeffrey VanWye, Hana Totary-Jain
mRNA therapeutics hold great promise for the treatment of human diseases. While incorporating naturally occurring modified nucleotides during synthesis has greatly increased their potency and safety, challenges in selective expression have hindered clinical applications. microRNA-regulated in vitro transcribed mRNAs, called microRNA switches, have been used to control expression of exogenous mRNA in a cell-selective manner. However, the effect of nucleotide modifications on microRNA-dependent silencing has not been examined. Here we show that incorporation of pseudouridine, N1-methylpseudourdine or pseudouridine/5-methylcytidine, which increases translation, tends to decrease the regulation of miRNA switches. Moreover, pseudouridine/5-methylcytidine modification enables one miRNA target site at the 3’ UTR to be as effective as four target sites. We also demonstrate that the effects of pseudouridine, pseudouridine/5-methylcytidine, and N1-methylpseudourdine modification are miRNA switch-specific and do not correlate with the proportion of modified nucleotides in the miRNA target site. Furthermore, modified microRNA switches containing seed complementary target sites are poorly regulated by microRNA. We also show that placing the miRNA target site in the 5’ UTR of the miRNA switch abolishes the effect of nucleotide modification on miRNA-dependent silencing. This work provides insights into the influence of nucleotide modifications on miRNA-dependent silencing and informs the design of optimal miRNA switches.
Silenced long non-coding RNA PVT1 increased the radiosensitivity of non-small cell lung cancer through the microRNA-424-5p/lncRNA PVT1/CARM1 signaling pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-15 Dong Wang, Yi Hu
Long non-coding RNAs take crucial part in tumorigenesis. This study aims to explore the role of, lncRNA PVT1 on the radiosensitivity of non-small cell lung cancer (NSCLC) via the microRNA (miR)-424-5p/lncRNA PVT1/CARM1 signaling pathway. Differentially expressed lncRNA related to NSCLC was filtrated. The interaction between PVT1 and CARM1 and effect of miR-424-5p on cell biological processes were tested. PVT1 was the most remarkable lncRNA that upregulated in NSCLC. CARM1 was co-expressed with lncRNA PVT1 and associated with NSCLC radiosensitivity. Both lncRNA PVT1 and CARM1 can competitively combine with miR-424-5p.PVT1 was a target gene of miR-424-5p. PVT1, CARM1, MMP-2, MMP-9 and Bcl-2 as well as cell proliferation, migration and invasion were decreased but miR-424-5p, Bax and apoptosis were increased after transfection with siRNA-PVT1 or overexpressed miR-424-5p. Our data suggest the regulatory role of miR-424-5p/lncRNA PVT1/CARM1 signaling pathway in NSCLC radiosensitivity, and siRNA-PVT1 and overexpressed miR-424-5p increases radiosensitivity of NSCLC.
Endometrial epithelial cell apoptosis is inhibited via a ciR8073-miR181a-neurotensis pathway during embryo implantation Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-14 Lei Zhang, Xiaorui Liu, Sicheng Che, Jiuzeng Cui, Xingna Ma, Xiaopeng An, Binyun Cao, Yuxuan Song
Development of the receptive endometrium (RE) from the pre-receptive endometrium (PE) is essential for embryo implantation, but its molecular mechanisms have not been fully understood. In this study, lncRNA/circRNA-miRNA-mRNA networks were constructed to explore the functions of potential competing endogenous RNAs (ceRNA) during the development of RE in dairy goats. We observed that circRNA8073 (ciR8073) decreased the levels of miR-181a by acting as a miRNA sponge. This effect indirectly increased the expression of neurotensin in endometrial epithelial cells (EECs). Neurotensin then inhibited EEC apoptosis by the increasing expression of BCL-2/BAX in favor of BCL-2, via the MAPK pathway, and also induced increased expression of leukemia-inhibitory factor, cyclo-oxygenase 2, vascular endothelial growth factor A, and homeobox A10. We have thus identified a ciR8073-miR181a-neurotensin pathway in the endometrium of dairy goats. Through this pathway, ciR8073 functions as a ceRNA that sequesters miR-181a, thereby protecting neurotensin transcripts from miR-181a-mediated suppression in EECs.
Optimizing synthetic miRNA minigene architecture for efficient miRNA hairpin concatenation and multi-target gene knockdown Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-14 Francis Rousset, Patrick Salmon, Simon Bredl, Ophélie Cherpin, Marta Coelho, Renier Myburgh, Marco Alessandrini, Michael Perny, Marta Roccio, Roberto F. Speck, Pascal Senn, Karl Heinz Krause
Synthetic miRNA minigenes, SMIG, have a major potential for molecular therapy, however their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here we investigate the overall architecture of SMIG driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared to constructs containing an intercalated sequence (“spacer”). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP–based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knockdown their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knock-down SMIG, demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22phox was achieved.
Activating PTEN tumor suppressor expression with the CRISPR/dCas9 system Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-14 Colette Moses, Fiona Nugent, Charlene Babra Waryah, Benjamin Garcia-Bloj, Alan R. Harvey, Pilar Blancafort
PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF-mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR and MAPK signaling. BRAF V600E-mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf or PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.
In silico prediction of small molecule-miRNA associations based on HeteSim algorithm Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-13 Jia Qu, Xing Chen, Ya-Zhou Sun, Yan Zhao, Shu-Bin Cai, Zhong Ming, Zhu-Hong You, Jian-Qiang Li
Targeting microRNAs (miRNAs) with drug small molecules (SM) is a new treatment method for many human complex diseases. Unsurprisingly, identification of potential miRNA-SM associations is helpful for pharmaceutical engineering and disease therapy in the field of medical research. In this paper, we developed a novel computational model of HeteSim based inference for Small Molecule-MiRNA Association prediction (HSSMMA) by implementing a path-based measure method of HeteSim on a heterogeneous network combined with known miRNA-SM associations, integrated miRNA similarity and integrated SM similarity. Through considering paths from a SM to a miRNA in the heterogeneous network, the model can capture the semantics information under each path and predict potential miRNA-SM associations based on all the considered paths. We performed global, miRNA-fixed local and SM-fixed local leave one out cross validation (LOOCV) as well as 5-fold cross validation based on the dataset of known miRNA-SM associations to evaluate the prediction performance of our approach. The results showed that HSSMMA gained the corresponding AUCs of 0.9913, 0.9902, 0.7989, 0.9910+/-0.0004 based on Dataset1 and AUCs of 0.7401, 0.8466, 0.6149, 0.7451+/-0.0054 based on Dataset 2, respectively. In case studies, 2 out of the top 10 and 13 out of the top 50 predicted potential miRNA-SM associations were confirmed by published literatures. We further implemented case studies to test whether HSSMMA was effective for new SMs without any known related miRNAs. The results from cross validation and case studies showed that HSSMMA could be a useful prediction tool for the identification of potential miRNA-SM associations.
The modulatory role of microRNA-873 in progression of KRAS-driven cancers Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-13 Hamada A. Mokhlis, Recep Bayraktar, Nashwa N. Kabil, Ayse Caner, Nermin Kahraman, Cristian Rodriguez-Aguayo, Erika P. Zambalde, Jianting Sheng, Kübra Karagoz, Pinar Kanlikilicer, Abdel Aziz H. Abdel Aziz, Tamer M. Abdelghany, Ahmed A. Ashour, Stephen Wong, Michael L. Gatza, George A. Calin, Gabriel Lopez-Berestein, Bulent Ozpolat
KRAS is one of the most frequently mutated proto-oncogenes in pancreatic ductal adenocarcinoma (PDAC) and aberrantly activated in triple-negative breast cancer (TNBC). Profound role of microRNAs (miRNAs) in the pathogenesis of human cancer is being uncovered including in cancer therapy. Using in silico prediction algorithms, we identified miR-873 as a potential regulator of KRAS and investigated its role in PDAC and TNBC. We found that reduced miR-873 expression is associated with shorter patient survival in both cancers. miR-873 expression is significantly repressed in PDAC and TNBC cell lines and inversely correlated with KRAS levels. We demonstrated that miR-873 directly binds to the 3’-untranslated region (3’-UTR) of KRAS mRNA and suppressed its expression. Notably, restoring miR-873 expression induced apoptosis and recapitulated the effects of KRAS inhibition on cell proliferation, colony formation, and invasion and suppressed the activity of ERK and PI3K/AKT, while overexpression of KRAS rescued the effects mediated by miR-873. Moreover, in vivo delivery of miR-873-nanoparticles inhibited KRAS expression and tumor growth in PDAC and TNBC tumor models. In conclusion, we provide the first evidence that miR-873 acts as a tumor suppressor by targeting KRAS and that miR-873–based gene therapy may be a therapeutic strategy in PDAC and TNBC.
Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-10 Shih-Han Ko, Yi-Jen Liau, Ya-Hui Chi, Mei-Ju Lai, Yu-Ping Chiang, Chun-Yi Lu, Luan-Yin Chang, Woan-Yuh Tarn, Li-Min Huang
The molecular chaperon MRJ (DNAJB6) exhibits two splice isoforms that have different roles in human viral infection, but the regulatory mechanism of MRJ isoform expression is yet unclear. In this study, we show that reduction of the polyadenylation factor CstF64 was correlated with the increase of the MRJ large isoform (MRJ-L) in human macrophages, and elucidate the mechanism underlying CstF64-modulated MRJ isoform expression. Moreover, we exploited an antisense strategy targeting MRJ-L for virus replication. A morpholino oligonucleotide complementary to the 5’ splice site of MRJ intron 8 downregulated MRJ-L expression and suppressed the replication of not only human immunodeficiency virus (HIV-1) but also respiratory syncytial virus (RSV). We demonstrated that downregulation of MRJ-L level reduced HIV-1 replication as well as the subgenomic mRNA and viral production of RSV. The present finding that two human health-threatening viruses take advantage of MRJ-L for infection suggests MRJ-L as a potential target for broad-spectrum antiviral strategy.
Programmable molecular scissors: Applications of a new tool for genome editing in biotech Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-06 Subbroto Kumar Saha, Forhad Karim Saikot, Md.Shahedur Rahman, Mohammad Abu HenaMostofa Jamal, S.M.Khaledur Rahman, S.M.Riazul Islam, Ki-Hyun Kim
Targeted genome editing is an advanced technique that enables precise modification of the nucleic acid sequences in a genome. Genome editing is typically performed using tools, such as molecular scissors, to cut a defined location in a specific gene. Genome editing has impacted various fields of biotechnology such as agriculture; biopharmaceutical production; studies on the structure, regulation, and function of the genome; and the creation of transgenic organisms and cell lines. Although genome editing is used frequently, it has several limitations. Here, we provide an overview of well-studied genome-editing nucleases, including single-stranded oligodeoxynucleotides (ssODNs), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases (CRISPR-CAS9). To this end, we describe the progress toward editable nuclease-based therapies and discuss the minimization of off-target mutagenesis. Future prospects of this challenging scientific field are also discussed.
microRNA-based prophylaxis in a mouse model of cirrhosis and liver cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-06 Elisa Callegari, Marco Domenicali, Ram Charan Shankaraiah, Lucilla D’Abundo, Paola Guerriero, Ferdinando Giannone, Maurizio Baldassarre, Cristian Bassi, Bahaeldin K. Elamin, Barbara Zagatti, Manuela Ferracin, Francesca Fornari, Giuseppe Altavilla, Stella Blandamura, Enrico Maria Silini, Laura Gramantieri, Silvia Sabbioni, Massimo Negrini
Most hepatocellular carcinomas (HCCs) arise in the context of chronic liver disease and/or cirrhosis. Thus, chemoprevention in individuals at risk represents an important, but yet unproven approach. In this study, we investigated the ability of miRNA-based molecules to prevent liver cancer development in a cirrhotic model. To this end, we developed a mouse model able to recapitulate the natural progression from fibrosis to HCC and then we tested the prophylactic activity of a microRNA-based approach in the model. The studies were carried out in the TG221 transgenic mouse, characterized by the overexpression of miR-221 in the liver and predisposed to the development of liver tumors. TG221 as well as wild type mice were exposed to the hepatotoxin carbon tetrachloride (CCl4) to induce chronic liver damage. All mice developed liver cirrhosis, but only TG221 mice developed nodular lesions in 100% of cases within 6 months of age. The spectrum of lesions ranged from dysplastic foci to carcinomas. To investigate miRNA-based prophylactic approaches, anti-miR-221 oligonucleotides or miR-199a-3p mimics were administered to TG221 CCl4-treated mice. Compared to control animals, a significant reduction of number, size and, most significantly, malignant phenotype of liver nodules was observed, thus demonstrating an important prophylactic action of miRNA-based molecules. In summary, in this study we not only reported a simple model of liver cancer in a cirrhotic background, but also provided evidence for a potential miRNA-based approach to reduce the risk of HCC development.
Posttranscriptional Regulation of 14q32 microRNAs by RNA Binding Proteins CIRBP and HADHB during Vascular Regeneration after Ischemia Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-06 Angela Downie Ruiz Velasco, Sabine M.J. Welten, Eveline A.C. Goossens, Paul H.A. Quax, Juri Rappsilber, Gracjan Michlewski, A.Yaël Nossent
After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the miR genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32 and we aimed to identify these RBPs. In order to identify proteins responsible for this posttranscriptional regulation we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of Cold Inducible RNA Binding Protein (CIRBP) and Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Beta (HADHB) to the precursors of late upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hind limb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329, but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB-/- 3T3 cells, which display reduced expression of miR-329 and miR-495, but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs.
Efficient knockdown and lack of passenger stand activity by Dicer-independent short hairpin RNAs expressed from RNA Polymerase-II-driven microRNA scaffolds Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-01 Erik Kaadt, Sidsel Alsing, Claudia Cecchi, Christian Kroun Damgaard, Thomas J. Corydon, Lars Aagaard
Expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (pol-III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support loss of passenger strand activity. We demonstrate that artificial pri-miR transcripts, expressed from pol-II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of pol-II promoters allows for controlled expression, while the inclusion of pri-miR sequences likely requires Drosha processing and as such mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and improved safety.
circARF3 alleviates mitophagy mediated inflammation by targeting miR-103/TRAF3 in mice adipose tissue Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-01 Zhenzhen Zhang, Tiantian Zhang, Ruonan Feng, Hongtao Huang, Tianyu Xia, Chao Sun
Adipose inflammation is an important cause for obesity-associated metabolic disorders, including insulin resistance and hypertension. Here we investigated that a circular RNA (circRNA), which we termed circARF3, acts as an endogenous miR-103 sponge to alleviate adipose inflammation by promoting mitophagy. On the other hand, miR-103 aggravated inflammation by inhibiting mitophagy, revealing that miR-103 acts as a positive regulator of adipose inflammation. Furthermore, we found that TRAF3 as a miR-103 downstream target to mediate the functions of miR-103 in adipose inflammation. Overexpressing TRAF3 attenuated miR-103 induced inflammation by accelerating mitophagy. Moreover, we identified that circARF3 blocked miR-103 effects, which resulted in an increase of TRAF3 expression. TRAF3 restrained NF-κB signaling pathway, heightened mitophagy and suppressed NLRP3 inflammasome activation ultimately. Our finding showed that circARF3 acts as an endogenous miR-103 sponge to inhibit mitophagy mediated adipose inflammation both in vitro and in vivo. These findings disclose a new regulatory pathway for adipose inflammation, which consists of circARF3, miR-103 and TRAF3. This study can be a useful addition to our knowledge as provides as a new strategy for the preventing of adipose inflammation in obesity disorder.
Identifying genomic variations in monozygotic twins discordant for autism spectrum disorder using whole-genome sequencing Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-01 Yan Huang, Yue Zhao, Ying Yi, Jiayue Xu, Xiaodan Li, Zhaomin Gao, Xiaolei Zhan, Jia Yu, Dong Wang, Shuang Liang, Lijie Wu
Autism spectrum disorder (ASD) presents a set of childhood neurodevelopmental disorders with impairments in social communication and restricted, repetitive, and stereotyped patterns of behavior. Here, based on the whole genome sequencing (WGS) data of three monozygotic twins discordant for ASD, we explored multiple patient-specific genetic variations and prioritized a list of ASD risk genes. Our results identified DVMT (Discordant Variation in Monozygotic Twin) observed in at least two twin pairs, including 14310 SNPs, 2425 InDels and 16735 CNVs, referring to a total of 2174 genes, and 37 of these were covered by all three types of variations. Gene ontology (GO) enrichment analysis of biological processes for 2174 genes showed that the majority of these genes were related to neurodevelopmental processes. In addition to, functional network analysis showed that there was a strong functional relevance between 37 genes covered by all three types of variations. In conclusion, for the first time, we conducted a comprehensive scan of genomic differences between monozygotic twins discordant for ASD, providing researchers with in-depth directions. It may also provide effective strategies for clinical treatment of individuals affected by ASD.
An RNA aptamer targeting the receptor tyrosine kinase PDGFRα induces anti-tumor effects through STAT3 and p53 in glioblastoma (GBM) Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-12-01 Sorah Yoon, Xiwei Wu, Brian Armstrong, Nagy Habib, John J. Rossi
Human glioblastoma (GBM) is the most aggressive malignancy of the central nervous system, with less than 5% survival. Despite great efforts to find effective therapeutics, current options remain very limited. To develop a targeted cancer therapeutic, we selected RNA aptamers against platelet-derived growth factor receptor α (PDGFRα), which is a receptor tyrosine kinase. One RNA aptamer (PDR3) with high affinity (0.25 nM) showed PDGFRα specificity and was internalized in U251-MG cells. Following treatment with the PDR3 aptamer, expression of the transcription factor STAT3 was inhibited, while the expression of the histone demethylase JMJD3 and the tumor suppressor p53 were upregulated. PDR3 also upregulated serine phosphorylation of p53, which subsequently mediated apoptosis through the death receptors TRAIL-R1/R2, FADD, and Fas. PDR3 significantly decreased cell viability in a dose-dependent manner. Furthermore, translocation of PDR3 into the nucleus induced hypomethylation at the promoters of cyclin D2. To assess the feasibility of targeted delivery, we conjugated PDR3 aptamer with STAT3-siRNA for a chimera. The PDR3-siSTAT3 chimera successfully inhibited the expression of target genes and showed significant inhibition of cell viability. In summary, our results show that well-tailored RNA aptamers targeting the PDGFRα-STAT3 axis have the potential to act as anti-cancer therapeutics in GBM.
Genetic Alphabet Expansion Provides Versatile Specificities and Activities of Unnatural-base DNA Aptamers Targeting Cancer Cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-29 Kazunobu Futami, Michiko Kimoto, Yun Wei Shermane Lim, Ichiro Hirao
The potential of genetic alphabet expansion technologies using artificial extra base pairs (unnatural base pairs) has been rapidly expanding and increasing. We present that the hydrophobic unnatural base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which acts as a fifth letter in a DNA library, provides a series of high-affinity DNA aptamers with versatile binding specificities and activities to cancer cells. These Ds-containing DNA aptamers were generated by a method called cell-ExSELEX, to target three breast cancer cell lines, MCF7, MDA-MB-231, and T-47D. Aptamer 14A-MCF7, which targets MCF7 cells, specifically binds to MCF7 cells, but not other cancer cell lines. Aptamer 07-MB231, which targets MDA-MB-231 cells, binds to a series of metastatic bone and lung cancer cell lines. Aptamer 05-MB231 targets MDA-MB-231 cells, but it also binds to all of the cancer and leukemia cell lines that we examined. None of these aptamers bind to normal cell lines, such as MCF10A and HUVEC. In addition, aptamers 14A-MCF7 and 05-MB231 are internalized within the cancer cells, and aptamer 05-MB231 possesses anti-proliferative properties against most cancer cell lines that we examined. These aptamers and the generation method are broadly applicable to cancer cell imaging, biomarker discovery, cancer cell profiling, anti-cancer therapies, and drug delivery systems.
γδ T Cell-derived Extracellular Vesicles: an Efficient Delivery System for miR-138 with Anti-tumoral and Immunostimulatory roles on Oral Squamous Cell Carcinoma Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-24 Ling Li, Shun Lu, Xinhua Liang, Bangrong Cao, Shaoxin Wang, Jian Jiang, Huaichao Luo, Shuya He, Jinyi Lang, Guiquan Zhu
In this study, we sought to investigate the potential application of γδ T cell-derived extracellular vesicles (γδTDEs) as drug delivery system (DDS) for miR-138 in the treatment of oral squamous cell carcinoma (OSCC). Our data showed that overexpression of miR-138 in γδ T cells obtained miR-138-rich γδTDEs accompanying increased expansion and cytotoxicity of γδ T cells. γδTDEs inherited the cytotoxic profile of γδ T cells and could efficiently deliver miR-138 to OSCC cells, resulting an synergetic inhibition on OSCC both in vitro and in in vivo. The pre-immunization by miR-138-rich γδTDEs inhibited the growth of OSCC tumors in immunocompetent C3H mice but not in nude mice, suggesting an immunomodulatory role by miR-13-rich γδTDEs. γδTDEs and miR-138 additively increased the proliferation, IFN-γ production, and cytotoxicity of CD8+ T cells against OSCC cells. Only delivered by γδTDEs can miR-138 efficiently target PD-1 and CTLA-4 in CD8+ T cells. We conclude that γδTDEs delivering miR-138 could achieve synergetic therapeutic effects on OSCC, which is benefited from the individual direct anti-tumoral effects on OSCC and immunostimulatory effects on T cells by both γδTDEs and miR-138; γδTDEs could serve as an efficient drug delivery system for miRNAs in the treatment of cancer.
miR675 accelerates malignant transformation of mesenchymal stem cells by blocking DNA mismatch repair Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-24 Yanan Lu, Shuting Song, Xiaoxue Jiang, Qiuyu Meng, Chen Wang, Xiaonan Li, Yuxin Yang, Xiaoru Xin, Qidi Zheng, Liyan Wang, Hu Pu, Xin Gui, Tianming Li, Dongdong Lu
miR675 is highly expressed in several human tumor tissues and positively regulates cell progression. Herein, we demonstrate miR675 promotes malignant transformation of human mesenchymal stem cells. Mechanistically, we reveal miR675 enhances the expression of the polyubiquitin-binding protein p62. Intriguingly, P62 competes with SETD2 to bind histone H3 and then significantly reduces SETD2 binding capacity to substrate histone H3, triggering drastically reduction of three methylation on histone H3 thirty-sixth lysine(H3K36me3). Thereby, the H3K36me3-hMSH6-SKP2 triplex complex is significantly decreased. Notably, the ternary complex’s occupancy capacity on chromosome is absolutely reduced, preventing from DNA damage repair. In virtue of the reductive degradation ability of SKP2 for aging histone H3.3 bound to mismatch DNA, the aging histone H3.3 repair is delayed. Therefore, the mismatch DNA escapes to repair can trigger abnormal expression of several cell cycle related-genes and then causes the malignant transformation of mesenchymal stem cells. These observations strongly suggest understanding the novel functions of miR675 will help in the development of novel therapeutic approaches in a broad range of cancer types.
Proximity ligation assays for in situ detection of innate immune activation: focus on in vitro transcribed mRNA Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Emmeline L. Blanchard, Kristin H. Loomis, Sushma M. Bhosle, Daryll Vanover, Patrick Baumhof, Bruno Pitard, Chiara Zurla, Philip J. Santangelo
The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for isolation of individual pathways, do not provide kinetic activation or spatial information within tissues and cannot be translated into clinical studies. Here, we demonstrated the use of proximity ligation assays (PLA) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA’s sensitivity and specificity using well characterized soluble agonists. Next, we characterized PRR activation from in vitro transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA IM injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.
Macrophage-derived miRNA-containing exosomes induce peritendinous fibrosis after tendon injury through miR-21-5p/SMAD7 pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Haomin Cui, Yu He, Shuai Chen, Deming Zhang, Yaling Yu, Cunyi Fan
Following tendon injury, development of fibrotic healing response impairs tendon function and restricts tendon motion. Peritendinous tissue fibrosis poses a major clinical problem in hand surgery. The communication between macrophages and tendon cells has a critical role in regulating tendon healing process. Yet, the mechanisms macrophages use to control peritendinous fibrosis are not fully understood. Here, we analyze the role of macrophages in tendon adhesion in mice by pharmacological depleting them. Such macrophage-depleted mice have less peritendinous fibrosis formation around the injured tendon compared with wild-type littermates. Macrophage-depleted mice restart fibrotic tendon healing by treatment with bone marrow macrophage-derived exosomes. We show bone marrow macrophages secrete exosomal miR-21-5p that directly targets SMAD7, leading to activation of fibrogenesis in tendon cells. These results demonstrate intercellular crosstalk between bone marrow macrophages and tendon cells is mediated by macrophages-derived miR-21-5p containing exosomes that control fibrotic healing response, providing potential targets for prevention and treatment of tendon adhesion.
Antisense oligonucleotides targeting angiogenic factors as potential cancer therapeutics Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Bao T. Le, Prithi Raguraman, Tamer R. Kosbar, Susan Fletcher, Steve D. Wilton, Rakesh N. Veedu
Cancer is one of the leading causes of death worldwide and conventional cancer therapies such as surgery, chemotherapy and radiotherapy do not address the underlying molecular pathologies, leading to inadequate treatment and tumour recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-β, TGF-α, VEGF, Endoglin and Angiopoietins play important roles in regulating tumour development and metastasis, and serve as potential targets for developing cancer therapeutics. Nucleic acid-based therapeutic strategies have received significant attention in the last two decades, and antisense oligonucleotide-mediated intervention is a prominent therapeutic approach for targeted manipulation of gene expression. Clinical benefits of antisense oligonucleotides have been recognised by the US Food and Drug Administration, with full or conditional approval of Vitravene, Kynamro, Exondys51 and Spinraza. Herein, we review the scope of antisense oligonucleotides that target angiogenic factors towards tackling solid cancers.
An analysis of the expression and association with immune cell infiltration of the cGAS/STING pathway in pan-cancer Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Xiang An, Yuanyuan Zhu, Tongsen Zheng, Guangyu Wang, Minghui Zhang, Jiade Li, Hongbo Ji, Shijun Li, Shucai Yang, Dandan Xu, Zhiwei Li, Tianzhen Wang, Yan He, Lei Zhang, Weiwei Yang, Ran Zhao, Dapeng Hao, Xiaobo Li
Recent evidence shows that cGAS/STING signaling is essential for antitumor immunity by inducing the production of type I interferon and thus activating both innate and adaptive immunity based on gene knockout mouse models. However, the extensive detection of the expression of cGAS/STING signaling in human cancer and mining the roles of this signaling pathway in human cancer immunity has not been performed until now. In this study, we revealed that four key molecules (cGAS, STING, TBK1 and IRF3) in the cGAS/STING signaling is highly expressed in cancer tissues and the expression level of these genes is negatively correlated with their methylation levels in most of the detected cancer types. We also showed that highly upregulated cGAS/STING signaling is negatively correlated with the infiltration of immune cells in some tumor types, and consistent with these findings, we showed that a high level of cGAS/STING signaling predicts a poor prognosis in patients with certain cancers. This study suggests that it is necessary to deeply and fully evaluate the function of cGAS/STING signaling in cancer immunity and cancer progression before the application of the STING agonist-based anticancer immune therapy in the clinic.
A simple cloning-free method to efficiently induce gene expression using CRISPR/Cas9 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Lyujie Fang, Sandy S.C. Hung, Jennifer Yek, Layal El Wazan, Tu Nguyen, Shahnaz Khan, Shiang Y. Lim, Alex W. Hewitt, Raymond C.B. Wong
Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here we report a simple, cloning-free method to induce gene expression at endogenous locus using CRISPR/Cas9 activators. Our strategy utilises synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65 and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1 and NEUROD1) in human cells, whereas the rat cells exhibit a more variable and less efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular and cell biology disciplines.
GalNAc conjugation attenuates the cytotoxicity of antisense oligonucleotide drugs in renal tubular cells Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Sabine Sewing, Marcel Gubler, Régine Gérard, Blandine Avignon, Yasmin Mueller, Annamaria Braendli-Baiocco, Marielle Odin, Annie Moisan
Targeted delivery of antisense oligonucleotide (AON) drugs is a promising strategy to increase their concentration in the desired tissues and cell types while reducing access to other organs. Conjugation of AONs to N-acetylgalactosamine (GalNAc) has been shown to efficiently shift their biodistribution toward the liver via high affinity binding to the asialoglycoprotein receptor (ASGPR) expressed at the surface of hepatocytes. Nevertheless, GalNAc conjugation does not prevent accumulation of AONs in the kidney cortex and GalNAc-conjugated AONs might cause kidney toxicities, for example under conditions of ASGPR saturation. Here, we investigated the nephrotoxicity potential of GalNAc-conjugated AONs by in vitro profiling of AON libraries in renal proximal tubule epithelial cells (PTEC) and in vivo testing of selected candidates. Whereas GalNAc-conjugated AONs appeared generally innocuous to PTECs, some caused mild to moderate nephrotoxicity in rats. Interestingly, the in vivo kidney liabilities could be recapitulated in vitro by treating PTEC with the unconjugated (or naked) parental AONs. An in vitro mechanistic study revealed that GalNAc conjugation attenuated AON-induced renal cell toxicity despite intracellular accumulation similar to naked AONs and independent of target knock down. Overall, our in vitro findings reveal ASGPR-independent properties of GalNAc AONs that confer a favorable safety profile at the cellular level, which may variably translate in vivo due to catabolic transformation of circulating AONs.
MiRNA-31-5p mediates the proliferation and apoptosis of human spermatogonial stem cells via targeting JAZF1 and Cyclin A2 Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-20 Hongyong Fu, Fan Zhou, Qingqing Yuan, Wenhui Zhang, Qianqian Qiu, Xing Yu, Zuping He
Several lines of evidence highlight the important application of human spermatogonial stem cells (SSCs) in translational medicine. The fate decisions of SSCs are mainly mediated by genetic and epigenetic factors. We have recently demonstrated that PAK1 regulates the proliferation, DNA synthesis and early apoptosis of human SSCs through PDK1/KDR/ZNF367 and ERK1/2 and AKT pathway. However the underlying epigenetic mechanism of PAK1 in human SSCs remains unknown. In this study, we found that the level of miRNA-31-5p was elevated by PAK1 knockdown. CCK-8, PCNA and EDU assays revealed that miRNA-31-5p mimics inhibited cell proliferation and DNA synthesis of human SSCs. Annexin V/PI staining and flow cytometry showed that miRNA-31-5p increased the early and late apoptosis of human SSCs. Furthermore, JAZF1 was predicted and verified as a target of miRNA-31-5p, and the three-dimensional (3-D) structure model of JAZF1 protein was illustrated. JAZF1 silencing led to a reduction of cell proliferation and DNA synthesis as well as an enhancement of the early and late apoptosis of human SSCs. Finally, miRNA-31-5p mimics decreased the level of including Cyclin A2 rather than Cyclin D1 or Cyclin E1, and JAZF1 knockdown led to the reduction of Cyclin A2 in human SSCs. Collectively, miRNA-31-5p regulates the proliferation, DNA synthesis and apoptosis of human SSCs by the PAK1/JAZF1/Cyclin A2 pathway. This study thus offers a novel insight into the molecular mechanisms underlying the fate decisions of human SSCs and might provide novel targets for molecular therapy of male infertility.
A chymase inhibitory RNA aptamer improves cardiac function and survival after myocardial infarction Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-13 Denan Jin, Shinji Takai, Yosuke Nonaka, Satoko Yamazaki, Masatoshi Fujiwara, Yoshikazu Nakamura
We have reported that mast cell chymase, an angiotensin II-generating enzyme, is important in cardiovascular tissues. Recently, we developed a new chymase-specific inhibitory RNA aptamer, HA28, and evaluated the effects of HA28 on cardiac function and the mortality rate after myocardial infarction. Eechocardiographic parameters, such as left ventricular ejection fraction, fractional shortening, and the ratio of early to late ventricular filling velocities were significantly improved by treatment with HA28 after myocardial infarction. The mortality rate was significantly reduced in the HA28-treated group. Cardiac chymase activity and chymase gene expression were significantly higher in the vehicle-treated myocardial infarction group, and these were markedly suppressed in the HA28-treated myocardial infarction group. The present study provides the first evidence that a single-stranded RNA aptamer that is a chymase-specific inhibitor is very effective in the treatment of acute heart failure caused by myocardial infarction. Chymase may be a new therapeutic target in post myocardial infarction pathophysiology.
MicroRNA-29b Mediates Lung Mesenchymal-Epithelial Transition and Prevents Lung Fibrosis in the Silicosis Model Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-06 Jingping Sun, Qiuyue Li, Ximeng Lian, Zhonghui Zhu, Xiaowei Chen, Wanying Pei, Siling Li, Ali Abbas, Yan Wang, Lin Tian
A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-11-01 Zongliang Gao, Elena Herrera-Carrillo, Ben Berkhout
The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease. The large size and relative complexity of such CRISPR transgene cassettes impede their broad implementation, especially in gene therapy applications with viral vectors that have a limited packaging capacity. Here, we report the design of a single promoter driven CRISPR-Cas9 system that employs the dual-polymerase (II and III) activity of the H1 promoter. This size reduction strategy of the vector insert provides a significant titer advantage in the lentiviral vector (LV) over the regular CRISPR system.
Overcoming the undesirable CRISPR-Cas9 expression in gene correction Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-30 Emily Xia, Rongqi Duan, Fushan Shi, Kyle E. Seigel, Hartmut Grasemann, Jim Hu
The CRISPR-Cas9 system is attractive for gene therapy as it allows for permanent genetic correction. However, as a new technology, Cas9 gene editing in clinical applications faces major challenges, such as safe delivery and gene targeting efficiency. Cas9 is a foreign protein to recipient cells, thus its expression may prompt the immune system to eliminate gene-edited cells. To overcome these challenges, we have engineered a novel delivery system based on the helper-dependent adenoviral (HD-Ad) vector, which is capable of delivering genes to airway basal stem cells in vivo. Using this system, we demonstrate the successful co-delivery of both CRISPR-Cas9/single-guide RNA and the LacZ reporter or CFTR gene as donor DNA to cultured cells. HD-Ad vector genome integrity is compromised following donor DNA integration, and because the CRISPR-Cas9/single-guide RNA and donor DNA are carried on the same vector, CRISPR-Cas9 expression is concurrently eliminated. Thus, we show the feasibility of site-specific gene targeting with limited Cas9 expression. In addition, we achieve stable CFTR expression and functional correction in cultured cells following successful gene integration.
“The AT interstrand crosslink: structure, electronic properties and influence on charge transfer in ds-DNA” Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-26 Boleslaw T. Karwowski
Circular RNA transcriptomics analysis of primary human brain microvascular endothelial cells infected with meningitic Escherichia coli Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-26 Ruicheng Yang, Bojie Xu, Bo Yang, Jiyang Fu, Lu Liu, Nouman Amjad, Aoling Cai, Chen Tan, Huanchun Chen, Xiangru Wang
With their essential regulatory roles in gene expression and high abundance in the brain, circular RNAs (circRNAs) have recently attracted considerable attention. Many studies have shown that circRNAs play important roles in the pathology of central nervous system (CNS) diseases, but whether circRNAs participate in E. coli-induced bacterial meningitis is unclear. We used high-throughput sequencing to analyze the transcriptional profiles of host circRNAs in primary brain microvascular endothelial cells in response to meningitic E. coli. A total of 308 circRNAs were significantly altered, including 140 upregulated and 168 downregulated ones (p<0.05). KEGG and Gene Ontology enrichment of the parental genes of these altered circRNAs indicated that they are likely to be involved in diverse biological processes via influencing the expression of their parental genes. Coupled with our previous mRNA and microRNA sequencing data, a competing endogenous RNA analysis was performed and the potential regulatory network was preliminary constructed and validated. By revealing the transcriptional profiles of the host circRNAs involved in E. coli meningitis, it is envisaged that the novel insight gained into the regulatory mechanisms of circRNAs in the development of bacterial meningitis will lead to better understanding of how to prevent and treat bacterial CNS infections.
Hsa-miR-202-3p regulates Sertoli cell proliferation, synthesis function and apoptosis by targeting LRP6 and Cyclin D1 of Wnt/β-catenin signaling pathway Mol. Ther. Nucl. Acids (IF 5.66) Pub Date : 2018-10-25 Chao Yang, Chencheng Yao, Ruhui Tian, Zijue Zhu, Liangyu Zhao, Peng Li, Huixing Chen, Yuhua Huang, Erlei Zhi, Yuehua Gong, Yunjing Xue, Hong Wang, Qingqing Yuan, Zuping He, Zheng Li
MicroRNAs (miRNAs) play important roles in mammalian spermatogenesis which is highly dependent on Sertoli cells. However, the functions and mechanisms of miRNAs in regulating human Sertoli cells remain largely unknown. Here, we report that hsa-miR-202-3p mediates the proliferation, apoptosis and synthesis function of human Sertoli cells. MiR-202-3p was up-regulated in Sertoli cells of Sertoli-cell-only syndrome (SCOS) patients compared with obstructive azoospermia (OA) patients with normal spermatogenesis. Overexpression of miR-202-3p induced Sertoli cell apoptosis and inhibited cell proliferation and synthesis, and the effects were opposite when miR-202-3p was knockdown. LRP6 and Cyclin D1 of Wnt/β-catenin signaling pathway were identified as direct targets of miR-202-3p in Sertoli cells, which were validated by bioinformatics tools and dual luciferase reporter assay. Differentially expressed LRP6 and Cyclin D1 between OA and SCOS Sertoli cells were also verified. LRP6 siRNA inference not only mimiced the effects of miR-202-3p overexpression, but also antagonized the effects of miR-202-3p inhibition on Sertoli cells. Collectively, miR-202-3p control the proliferation, apoptosis and synthesis function of human Sertoli cells via targeting LRP6 and Cyclin D1 of Wnt/β-catenin signaling pathway. This study thus provides a novel insight into fate determinations of human Sertoli cells and niche of human testis.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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