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  • Dose-finding study and pharmacokinetics profile of the novel 13-mer antisense miR-221 inhibitor in Sprague Dawley rats
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-08
    Maria Teresa Di Martino; Mariamena Arbitrio; Daniele Caracciolo; Francesca Scionti; Pierosandro Tagliaferri; Pierfrancesco Tassone

    MiR-221 is overexpressed in several malignancies where it promotes tumor growth and survival by interfering with gene transcripts, including p27Kip1, PUMA, PTEN, and p57Kip2. We previously demonstrated that a novel 13-mer miR-221 inhibitor (LNA-i-miR-221) exerts antitumor activity against human cancer with a pilot favorable pharmacokinetics and safety profile in mice and non-naive monkeys. We here report a non-GLP/GLP dose-finding investigation of LNA-i-miR-221 in Sprague-Dawley rats. The safety of the intravenous dose (125 mg/kg/day) for four consecutive days, two treatment cycles, was investigated by a first non-GLP study. Toxicokinetics profile of LNA-i-miR-221 was next explored in a GLP study at three different doses (5, 12.5 and 125 mg/kg/day). Slight changes in blood parameters and histological findings in kidney were observed at the highest dose. These effects were reversible and consistent with in vivo ASO class effect. The NOAEL was established at 5 mg/kg/day. The plasma exposure of LNA-i-miR-221, based on C0 and AUC, suggested no differential sex effect. Slight accumulation occurred between cycles one and two but was not observed after four consecutive administrations. Taken together our findings demonstrate a safety profile of LNA-i-miR-221 in Sprague-Dawley rats and provide a reference translational framework and path for the development of other LNA miR inhibitors in Phase I clinical study.

    更新日期:2020-02-10
  • The processing, gene regulation, biological functions and clinical relevance of N4-acetylcytidine on RNA: A systematic review
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-08
    Gehui Jin; Mingqing Xu; Mengsha Zou; Shiwei Duan

    N4-acetylcytidine (ac4C) is often considered to be a conservative chemically modified nucleoside present on tRNA and rRNA. Recent studies have shown extensive ac4C modifications in human and yeast mRNAs. ac4C helps to correctly read codons during translation and improves translation efficiency and the stability of mRNA. At present, the research of ac4C involves a variety of detection methods. The formation of ac4C is closely related to NAT10 and its helpers such as TAN1 for tRNA ac4C and snoRNA for rRNA ac4C. Also, ac4C is associated with the development, progression, and prognosis of a variety of human diseases. Here we summarize the history of ac4C research and the detection technologies of ac4C. We then summarized the role and mechanism of ac4C in gene expression regulation and demonstrated the relevance of ac4C to a variety of human diseases, especially cancer. Finally, we list the future challenges of the ac4C research and demonstrate a research strategy for the interactions among several abundant modified nucleosides on mRNA.

    更新日期:2020-02-10
  • Targeting circulating SINEs and LINEs with DNase I provides metastases inhibition in experimental tumor models
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-08
    L.A. Alekseeva; A.V. Sen’kova; M.A. Zenkova; N.L. Mironova

    Tumor-associated cell-free DNAs (cfDNAs) are found to play some important roles at different stages of tumor progression; they are seems to be involved in the transformation of normal cells and contribute to tumor migration and invasion. DNase I is considered a promising cancer cure, due to its ability to degrade cfDNAs. Previous studies using murine tumor models have proved the high anti-metastatic potential of DNase I. Later circulating cfDNAs, especially tandem repeats associated with SINE and LINE elements, have been found to be the enzyme’s main molecular targets. Here, using Lewis lung carcinoma, melanoma B16 and lymphosarcoma RLS40 murine tumor models, we reveal that tumor progression is accompanied by an increase in the level of SINE and LINE elements in the pool of circulating cfDNAs. Treatment with DNase I decreased in the number and area of metastases by factor 3 - 10, and the size of the primary tumor node by factor 1.5 - 2, which correlated with 5-10-fold decreasing SINEs and LINEs. We demonstrated that SINEs and LINEs from cfDNA of tumor-bearing mice are able to penetrate human cells. The results indicate SINEs and LINEs could be important players in metastasis, what allows them to be considered as attractive new targets for anticancer therapy.

    更新日期:2020-02-10
  • Aptamer-functionalized drug-nanocarrier improves hepatocellular carcinoma towards normal by targeting neoplastic hepatocytes
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-05
    Samrat Chakraborty; ZewduYilma Dile; Somdyuti Chakraborty; Somdatta Roy; Biswajit Mukherjee; Shila Elizabeth Besra; Saikat Dewanjee; Alankar Mukherjee; Probir Kumar Ojha; Vinay Kumar; Ramkrishna Sen
    更新日期:2020-02-06
  • CRISPR-Cas13a cleavage of dengue virus NS3 gene efficiently inhibits viral replication
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-05
    Hao Li; Shan Wang; Xue Dong; Qiao Li; Min Li; Junfeng Li; Yan Guo; Xia Jin; Yusen Zhou; Hongbin Song; Zhihua Kou

    CRISPR-Cas9 system has been applied to DNA editing with precision in eukaryotic and prokaryotic system, but it is unable to edit RNA directly. A recently developed CRISPR-Cas13a system has been shown to be capable of effectively knockdown RNA expression in mammalian and plant cells. Here, we employ CRISPR-Cas13a system to achieve reprogrammable inactivation of dengue virus in mammalian cells. Quantitative RT-PCR, FACS and plaque assay showed that crRNA targeting NS3 region led to the greatest viral inhibition among 10 crRNAs targeting different regions along the dengue viral genomic RNA. Deletions and insertions had also been found adjacent to the NS3 region after NS3-crRNA/Cas13a complex transfection. Our results demonstrate that CRISPR-Cas13a system is a novel and effective technology to inhibit dengue viral replication, suggesting that such a programmable method may be further developed into a novel therapeutic strategy for dengue and other RNA viruses.

    更新日期:2020-02-06
  • METTL3 facilitates oral squamous cell carcinoma tumorigenesis by enhancing c-Myc stability via YTHDF1-mediated m6A modification
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-04
    Wei Zhao; Yameng Cui; Lina Liu; Xiaozhou Ma; Xiaoqian Qi; Yue Wang; Zihao Liu; Shiqing Ma; Jingwen Liu; Jie Wu

    N6-Methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) occured on the N6 nitrogen of adenosine. However, the roles of m6A in oral squamous cell carcinoma (OSCC) are still elusive. Here, we investigate the function and mechanism of methyltransferase like 3 (METTL3) in the OSCC tumorigenesis. Clinically, METTL3 was significantly up-regulated in tissue samples and correlated with the poor prognosis of OSCC patients. Functionally, loss and gain studies illustrated that METTL3 promoted the proliferation, invasion and migration of OSCC cells in vitro, and METTL3 knockdown inhibited tumor growth in vivo. Mechanistically, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that METTL3 targeted the 3’-UTR (near to stop codon) of c-Myc transcript to install the m6A modification, thereby enhancing its stability. Furthermore, results revealed that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) mediated the m6A-increased stability of c-Myc mRNA catalyzed by METTL3. In conclusion, our findings herein identify that METTL3 accelerates the c-Myc stability via YTHDF1-mediated m6A modification, thereby giving rise to OSCC tumorigenesis.

    更新日期:2020-02-06
  • LncRNA KCNQ1OT1 suppresses the inflammation and proliferation of vascular smooth muscle cells through IκBa in intimal hyperplasia
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-04
    Bozhi Ye; Zi-heng Wu; Tung Yu Tsui; Bao-fu Zhang; Xiang Su; Yi-hui Qiu; Xiang-tao Zheng

    Inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the key events in intimal hyperplasia. This study aimed to explore the mechanism by which lncRNA KCNQ1OT1 affects VSMC inflammation and proliferation in this context. A vein graft model was established in mice to introduce intimal hyperplasia. Isolated normal VSMCs were induced with platelet-derived growth factor type BB (PDGF-BB), and the cell proliferation, migration, and secretion of inflammatory factors were determined. The results showed that KCNQ1OT1 was downregulated in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-treated VSMCs, and such downregulation of KCNQ1OT1 resulted from the increased methylation level in the KCNQ1OT1 promoter. Overexpressing KCNQ1OT1 suppressed PDFG-BB-induced VSMC proliferation, migration, and secretion of inflammatory factors. In VSMCs, KCNQ1OT1 bound to the IκBa protein and increased the cellular IκBa level by reducing phosphorylation and promoting ubiquitination of the IκBa protein. Meanwhile, KCNQ1OT1 promoted the expression of IκBa by sponging miR-221. The effects of KCNQ1OT1 knockdown on promoting VSMC proliferation, migration, and secretion of inflammatory factors were abolished by IκBa overexpression. The roles of KCNQ1OT1 in reducing the intimal area and inhibiting IκBa expression were proved in the vein graft mouse model after KCNQ1OT1 overexpression. In conclusion, KCNQ1OT1 attenuated intimal hyperplasia by suppressing the inflammation and proliferation of VSMCs, in which the mechanism upregulated IκBa expression by binding to the IκBa protein and sponging miR-221.

    更新日期:2020-02-06
  • Comprehensive analysis of RNA sequencing in adenoma-cancer transition identified predictive biomarkers and therapeutic targets of human colorectal cancer
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-04
    Mingzhe Zhu; Yanqi Dang; Zhenhua Yang; Yang Liu; Li Zhang; Yangxian Xu; Wenjun Zhou; Guang Ji

    Specific molecular biomarkers for predicting the transition from colorectal adenoma to cancer have been identified, however, circular RNA (circRNA) related signatures remain to be clarified. We carried out high-throughput RNA sequencing to determine the expression profiles of circRNAs, microRNAs (miRNAs) and mRNAs in human colorectal cancer (CRC), adenoma and adjacent normal tissues. We identified 84 circRNAs, 41miRNAs and 398 mRNAs that were commonly differentially expressed in CRC and adenoma tissues compared with normal tissues. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) analyses identified numerous cancer related hub genes that might serve as potential therapeutic targets in CRC. Competing endogenous RNA (ceRNA) networks including three circRNAs, three miRNAs and 28 mRNAs were constructed, suggesting their potential role in cancer progression. Representative differentially expressed RNAs were validated by the Cancer Genome Atlas (TCGA) database and Real-time PCR experiments. Receiver operating characteristic (ROC) curve analysis identified three circRNAs (hsa_circ_0049487, hsa_circ_0066875 and hsa_circ_0007444) as possible novel biomarkers predicting the transition from colonic adenoma to cancer. Overall, our findings may provide novel perspectives to clarify the mechanisms of the transition from premalignant adenoma to cancer, and identify specific circRNA related signatures with possible applications for the early diagnosis of and as potential therapeutic targets in CRC.

    更新日期:2020-02-04
  • The lncRNA-GAS5/miR-221-3p/DKK2 axis modulates the ABCB1-mediated adriamycin resistance of breast cancer via Wnt/β-catenin signal pathway
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-02-01
    Zhaolin Chen; Tingting Pan; Duochen Jiang; Le Jin; Yadi Geng; Xiaojun Feng; Aizong Shen; Lei Zhang
    更新日期:2020-02-03
  • sgRNA-PSM: predict sgRNAs on-target activity based on Position Specific Mismatch
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-31
    Bin Liu; Zhihua Luo; Juan He

    As a key technique for CRISPR-Cas9 system, identification of sgRNAs on-target activity is critical for both theoretical research (investigation of RNA functions) and real world applications (genome editing and synthetic biology). Because of its importance, several computational predictors have been proposed to predict the sgRNAs on-target activity. All these methods have obviously contributed to the developments of this very important field. However, they are suffering from certain limitations. We proposed two new protocols called “sgRNA-PSM” and “sgRNA-ExPSM” for sgRNAs on-target activity prediction via capturing the long-range sequence information and evolutionary information using a new way to reduce the dimension of the feature vector to avoid the risk of over-fitting. Rigorous leave-one-gene-out cross-validation on a benchmark dataset with 11 human genes and 6 mouse genes, and an independent dataset indicated that the two new protocols outperformed other competing methods. To make it easier for users to use the proposed sgRNA-PSM predictor, we have established a corresponding web server, which is available at http://bliulab.net/sgRNA-PSM/.

    更新日期:2020-01-31
  • A Dual functioning 5’PPP-NS1shRNA that activates RIG-I antiviral pathway and suppresses influenza nonstructural protein 1
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-31
    Neetu Singh; Priya Ranjan; Weiping Cao; Jenish Patel; Shivaprakash Gangappa; Bruce A. Davidson; John M. Sullivan; Paras N. Prasad; Paul R. Knight; Suryaprakash Sambhara

    Retinoic acid-inducible gene-I (RIG-I) is a cytosolic pathogen sensor which is crucial against a number of viral infections. Many viruses have evolved to inhibit pathogen sensors to suppress host innate immune responses. In the case of influenza, nonstructural protein 1 (NS1) suppresses RIG-I function leading to viral replication, morbidity and mortality. We show that silencing NS1 with in vitro transcribed 5’-triphosphate containing NS1 shRNA designed using the conserved region of a number of Influenza viruses not only prevented NS1 expression but also induced RIG-I activation and type I interferon expression resulting in an anti-viral state leading to inhibition of influenza virus replication in vitro. In addition, administration of 5’PPP-NS1 shRNA in prophylactic and therapeutic settings resulted in significant inhibition of viral replication following viral challenge in vivo in mice with corresponding increases of RIG-I, IFN β and λ, as well as a decrease in NS1 expression.

    更新日期:2020-01-31
  • Exosome-miR-155 derived from gastric carcinoma promotes angiogenesis by targeting c-MYB/VEGF axis of endothelial cells
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-30
    Ting Deng; Haiyang Zhang; Haiou Yang; Huiya Wang; Ming Bai; Wu Sun; Xinyi Wang; Yiran Si; Tao Ning; Le Zhang; Hongli Li; Shaohua Ge; Rui Liu; Dan Lin; Shuang Li; Guoguang Ying; Yi Ba

    Exosomes, membranous nanovesicles, are identified to naturally carry proteins, mRNAs, and microRNAs (miRNAs) and play important roles in tumor pathogenesis. Here, we showed that gastric cancer (GC) cell-derived exosomes can function as vehicles to deliver miR-155 to promote angiogenesis in GC. In this study, we first detected the expression of miR-155 and c-MYB was negatively correlated in GC and c-MYB was a direct target of miR-155. We next characterized the promotion effect of exosome-delivered miR-155 on angiogenesis and tumor growth in GC. We found that the miR-155 could inhibit c-MYB but increase vascular endothelial growth factor (VEGF) expression, and promote the growth, metastasis, and tube formation of vascular cells, as the reason of occurrence and development of tumors. We also used a tumor implantation mouse model to show that exosomes containing miR-155 significantly augment the growth rate of the vasculature and tumors in vivo. In a word, our results illustrated the potential mechanism between miR-155 and angiogenesis in GC. These findings contribute to our understanding of the functions of miR-155 and exosomes as a carrier for therapy of GC.

    更新日期:2020-01-31
  • Pan-cancer analysis reveals the diverse landscape of novel sense and antisense fusion transcripts
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-29
    Neetha Nanoth Vellichirammal; Abrar Albahrani; Jasjit K. Banwait; Nitish K. Mishra; You Li; Shrabasti Roychoudhury; Mathew J. Kling; Sameer Mirza; Kishor K. Bhakat; Vimla Band; Shantaram S. Joshi; Chittibabu Guda

    Gene fusions that contribute to oncogenicity can be explored for identifying cancer biomarkers and potential drug targets. To investigate the nature and distribution of fusion transcripts in cancer, we examined the transcriptome data of about 9,000 primary tumors from 33 different cancers in TCGA along with cell line data from CCLE using ChimeRScope, a novel fusion detection algorithm. We identified several fusions with sense (canonical- 39%) or antisense (non-canonical- 61%) transcripts recurrent across cancers. The majority of the recurrent non-canonical fusions found in our study are novel, unexplored and exhibited highly variable profiles across cancers, with breast cancer and glioblastoma having the highest and lowest rates, respectively. Overall, 4,344 recurrent fusions were identified from TCGA in this study, of which 70% were novel. Additional analysis of 802 tumor-derived cell line transcriptome data across 20 cancers revealed significant variability in recurrent fusion profiles between primary tumors and corresponding cell lines. A subset of canonical and non-canonical fusions was validated by examining the structural variation evidence in WGS data or by Sanger sequencing of fusion junctions. Several recurrent fusion genes identified in our study show promise for drug repurposing in basket trails and present opportunities for mechanistic studies.

    更新日期:2020-01-30
  • Tumor suppressive microRNA-216b binds to TPX2 activating the p53 signaling in human cutaneous squamous cell carcinoma
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-28
    Cheng Feng; Hai-Lin Zhang; Ang Zeng; Ming Bai; Xiao-Jun Wang

    Dysregulation of microRNAs (miRNAs) is acknowledged in human cutaneous squamous cell carcinoma (cSCC). We, hereby, evaluated the ability of microRNA-216b (miR-216b) to impact human cSCC. cSCC tissues with corresponding adjacent normal tissues were collected from 40 patients diagnosed with cSCC where the expression pattern of miR-216b and targeting protein for Xenopus kinesin-like protein 2 (TPX2) was determined by RT-qPCR and Western blot analysis. A431 cells were transfected with miR-216b mimic, miR-216b inhibitor, or short interfering RNA against TPX2 to evaluate cell proliferation, invasion, migration and apoptosis using MTT assay, scratch test, Transwell assay and flow cytometry. TPX2 was highly expressed in cSCC tissues while miR-216b was poorly expressed in association with tumor differentiation, lymph node metastasis and tumor node metastasis staging in patients with cSCC. In response to overexpressed miR-216b or silenced TPX2, cSCC cell proliferation, invasion, and migration were suppressed and apoptosis was stimulated, along with activated p53 signaling. Thus, upregulated miR-216b was capable of promoting apoptosis and inhibiting proliferation, invasion, and migration of cSCC cells by downregulating TPX2 through activation of the p53 signaling, highlighting a novel biomarker for novel treatment modalities against cSCC.

    更新日期:2020-01-30
  • Efficient delivery of antisense oligonucleotides using bioreducible lipid nanoparticles in vitro and in vivo
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-25
    Liu Yang; Feihe Ma; Fang Liu; Jinjin Chen; Xuewei Zhao; Qiaobing Xu
    更新日期:2020-01-26
  • Discovering Cancer-related miRNAs from miRNA-target Interactions by Support Vector Machines
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-25
    Cong Pian; Shanjun Mao; Jiali Yang; Guangle Zhang; Jin Du; Suet Yi Leung; Yuanyuan Chen; Xiaodan Fan

    miRNAs have been shown to be closely related to cancer progression. Traditional methods for discovering cancer-related miRNAs mostly require significant marginal differential expression, but some cancer-related miRNAs may be non-differentially or only weakly differentially expressed. Such miRNAs are called as Dark Matters miRNAs (DM-miRNAs) and targeted by Pian et al. (2018) through the Pearson correlation change on miRNA-target interactions (MTIs); but the efficiency of their method heavily relies on restrictive assumptions. In this paper, a novel method was developed to discover DM-miRNAs using Support Vector Machine (SVM) based on not only the miRNA expression data but also the expression of its regulating target. The application of the new method in breast and kidney cancer datasets found respectively 9 and 24 potential DM-miRNAs that cannot be detected by previous methods. 8 and 15 of the newly discovered miRNAs have been found to be associated with breast and kidney cancers respectively in existing literature, respectively. These results indicate that our new method is more effective in discovering cancer-related miRNAs.

    更新日期:2020-01-26
  • The identification of Long non-coding RNA H19 target and its function in Chronic myeloid leukemia (CML)
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-25
    Juhua Yang; Zhao Yin; Yumin Li; Yanjun Liu; Guiping Huang; Chunming Gu; Jia Fei

    H19 is a Long non-coding RNA which was lowly expressed in CML. Here,we found that the overexpression of H19 significantly inhibited cell viability and colony formation, and prolong survival in CML cell lines and three xenografted mouse models. The H19 target proteins and miRNAs were identified using a combination of computational prediction and RNA-Pull down, including PCBP1, FUS protein and miR-19a-3p, miR-106b-5p. Targeting PCBP1, FUS protein, miR-19a-3p and miR-106b-5p significantly inhibit the cell growth and colony formation of CML cell lines. Co-overexpression of H19 and PCBP1, FUS, miR-19a-3p and miR-106b-5p decrease the inhibitory effect of H19 in CML. These findings might provide a novel molecular insight into CML.

    更新日期:2020-01-26
  • Long Non-Coding RNA KLF3-AS1 Suppresses Cell Migration and Invasion in Esophageal Squamous Cell Carcinoma by Impairing miR-185-5p-targeted KLF3 Inhibition
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-25
    Jun-Qi Liu; Ming Deng; Nan-Nan Xue; Ting-Xuan Li; Yue-Xin Guo; Liang Gao; Di Zhao; Rui-Tai Fan

    Esophageal squamous cell carcinoma (ESCC) is a common cancer occurring to males and females worldwide. Accumulating evidence continues to highlight the crucial roles of long non-coding RNAs (lncRNAs) in the process of tumorigenesis. However, the regulatory mechanism of lncRNAs in ESCC remains unclear. The aim of this study is to elucidate the role of lncRNA KLF3-AS1 in ESCC by regulating miR-185-5p and KLF3. Initially, ESCC cell spheres with stem cell-like properties were prepared by suspension culture, and subsequently characterized by assessing colony formation ability and stem cell markers. LncRNA KLF3-AS1 was found to be poorly expressed in ESCC and could upregulate the expression of KLF3 by binding to miR-185-5p. LncRNA KLF3-AS1 upregulation was observed to inhibit miR-185-5p whereby contributing to decreased expression of SOX2 and Oct4. Furthermore, enhancement of lncRNA KLF3-AS1 resulted in reduced colony formation ability, cell invasion and migration and tumor volume in vivo while promoting cell apoptosis in ESCC through downregulation of miR-185-5p. Collectively, this study indicated that lncRNA KLF3-AS1 inhibited ESCC cell invasion and migration by impairing miR-185-5p-mediated inhibition of KLF3, highlighting a promising novel potential target for ESCC treatment.

    更新日期:2020-01-26
  • Knockdown of MCM3AP-AS1 Inhibits Proliferation, Invasion and Migration of Prostate Cancer Cells via DNMT1/DNMT3 (A/B) Methylation-Mediated Upregulation of NPY1R
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-23
    Xin Li; Jiancheng Lv; Shuai Liu

    Prostate cancer (PCa) is a heterogeneous tumor that commonly occurs among males worldwide. This study explored potential role long non-coding RNA MCM3AP-AS1 plays in PCa progression and investigated its mechanism. MCM3AP-AS1 and neuropeptide Y receptor Y1 (NPY1R) expression was determined in PCa cells. The regulatory of MCM3AP-AS1 in PCa cells was defined using scratch test, Transwell assay, EdU assay and flow cytometry. Methylation-specific PCR (MSP) was used to test the methylation level of NPY1R. Subsequently, the interaction among MCM3AP-AS1, DNA methyltransferase (DNMT)1/DNMT3 (A/B) and NPY1R was investigated using RNA immunoprecipitation, RNA pull-down and chromatin immunoprecipitation. Finally, we observed xenograft tumor in nude mice. MCM3AP-AS1 was highly while NPY1R was poorly expressed in PCa. Lentivirus-mediated overexpression of MCM3AP-AS1 promoted proliferation, invasion and migration while suppressing apoptosis of PCa cells, while opposite trends were detected after inhibition of MAPK pathway. MCM3AP-AS1 promoted methylation of NPY1R promoter via recruitment of DNMT1/DNMT3 (A/B), thereby downregulating NPY1R expression to activate the mitogen-activated protein kinase (MAPK) pathway. Furthermore, overexpressed MCM3AP-AS1 was observed to facilitate PCa development in vivo, which could be reversed by overexpressed NPY1R. Altogether, MCM3AP-AS1 silencing inhibits PCa progression by disrupting methylation of the NPY1R promoter to inactivate the MAPK pathway.

    更新日期:2020-01-23
  • MicroRNA-20b Promotes Cardiac Hypertrophy by the Inhibition of Mitofusin 2-Mediated Inter-organelle Ca2+ Cross-talk
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-23
    Yue Qiu; Rongchao Cheng; Chaoqi Liang; Yuan Yao; Wenhao Zhang; Jie Zhang; Mingyu Zhang; Baiyan Li; Chaoqian Xu; Rong Zhang

    MicroRNA and mitofusin-2 (Mfn2) are important in the development of cardiac hypertrophy, but the target relationship and mechanism associated with Ca2+ handling between SR and mitochondria under hypertrophic condition is not established. Mfn2 expression, Mfn2-mediated interorganelle Ca2+ cross-talk, and target regulation by miRNA-20b (miR-20b) were evaluated using animal/cellular hypertrophic models with state-of-art techniques. The results demonstrated that Mfn2 was down-regulated and miR-20b was up-regulated upon the target binding profile under hypertrophic condition. Our data showed that miR-20b induced cardiac hypertrophy that was reversed by rAAV9-anti-miR-20b or AMO-20b. The deleterious action of miR-20b on Mfn2 expression/function and mitochondrial ATP synthesis was observed and reversed by rAAV9-anti-miR-20b or AMO-20b. The targeted regulation of miR-20b on Mfn2 was confirmed by luciferase reporter and microRNA-masking. Importantly, the facts that mitochondrial calcium uniporter (MCU) activation by Spermine increased the cytosolic Ca2+ into mitochondria, manifested as enhanced histamine-mediated Ca2+ release from mitochondrial, suggesting that Ca2+ reuptake/buffering capability of mitochondria to cytosolic Ca2+ is injured by miR-20b-mediated Mfn2 signaling, by which leads cytosolic Ca2+ overload and cardiac hypertrophy through Ca2+ signaling pathway. In conclusion, pro-hypertonic miR-20b plays crucial roles in cardiac hypertrophy through down-regulation of Mfn2 and cytosolic Ca2+ overload by weakening the buffering capability of mitochondria.

    更新日期:2020-01-23
  • Down-regulation of hypoxia-inducible factor-1α by RNA interference alleviates the development of collagen-induced arthritis in rats
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-22
    Yiping Hu; Tiantian Zhang; Jingqin Chen; WenXiang Cheng; Jianhai Chen; Zhengtan Zheng; Jietao Lin; Guoyuan Zhu; Yong Zhang; Xueling Bai; Yan Wang; Bing Song; Qingwen Wang; Ling Qin; Peng Zhang

    RA is the most common type of autoimmune arthritis. HIF-1α as a transcription factor in response to hypoxia suggests that it could be a potential therapeutic target for the treatment of RA. In this study, we assessed whether HIF pathway blockade attenuates the manifestations of RA in the collagen-induced arthritis (CIA) rat model. We constructed a short hairpin RNA (shRNA) lentiviral expression vector targeting HIF-1α (pLVX-shRNA-HIF-1α) and to achieve HIF-1α RNA interference. Quantitative RT-PCR, immunofluorescence staining and Western blot were used to detect the expressions of HIF-1α, VEGF, p-p65 and p-IКBɑ mRNA and protein, respectively. Micro-Computed Tomography was used to investigate joint morphology at different time points after CIA induction. Moreover, ELISA was used to monitor the expression of inflammatory cytokines. In vitro analyses revealed that pLVX-shRNA-HIF-1α effectively inhibited the expression of HIF-1α and VEGF and led to the activation of p-65 and p-IКBɑ, as well as decreased pro-inflammatory cytokine expression in cell culture. Inhibition of HIF-1α in rats decreased signed of systemic inflammatory condition together with decreased pathological changes of RA. Moreover, downregulation of HIF-1α expression markedly reduced the synovitis and angiogenesis. In conclusion, we have shown that pharmacological inhibition of HIF-1 may improve the clinical manifestations of RA.

    更新日期:2020-01-22
  • VALIDATION OF MIR-20A AS A TUMOR SUPPRESSOR GENE IN HEPATOCELLULAR CARCINOMA USING HEPATOCYTE-SPECIFIC HYPERACTIVE PIGGYBAC TRANSPOSONS
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-22
    Tipanee Jaitip; Di Matteo Mario; Tulalamba Warut; Samara-Kuko Ermira; Keirsse Jiri; Van Ginderachter Jo A.; Chuah Marinee, Khim; Thierry VandenDriessche

    We established a semi-high throughput in vivo screening platform using hyperactive piggyBac (hyPB) transposons (designated as PB-miR) to identify microRNAs miRs that inhibit hepatocellular carcinoma (HCC) development in vivo, following miR overexpression in hepatocytes. PB-miRs encoding 6 different miRs from the miR-17-92b cluster and 9 miRs from outside this cluster were transfected into mouse livers that were chemically induced to develop HCC. In this slow-onset HCC model, miR-20a significantly inhibited HCC. Next, we developed a more aggressive HCC model by overexpression of HRASG12V and c-MYC oncogenes that accelerated HCC development after only 6 weeks. The tumor suppressor effect of miR-20a could be demonstrated even in this rapid-onset HRASG12V/c-MYC HCC model consistent with significantly prolonged survival and decreased HCC tumor burden. Comprehensive RNA expression profiling of 95 selected genes typically associated with HCC development revealed differentially expressed genes and functional pathways that were associated with miR-20a-mediated HCC suppression. To our knowledge, this is the first study establishing a direct causal relationship between miR-20a over-expression and liver cancer inhibition in vivo. Moreover, these results demonstrate that hepatocyte-specific hyPB transposons are an efficient platform to screen and identify miRs that affect overall survival and HCC tumor regression.

    更新日期:2020-01-22
  • miR-25 promotes cardiomyocyte proliferation by targeting FBXW7
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-21
    Bei Wang; Mengting Xu; Miaomiao Li; Fujian Wu; Shijun Hu; Xiangbo Chen; Liqun Zhao; Zheyong Huang; Feng Lan; Dong Liu; Yongming Wang

    Induction of endogenous cardiomyocyte (CM) proliferation is one of the key strategies for heart regeneration. Increasing evidence points to the potential role of microRNAs (miRNAs) in the regulation of CM proliferation. Here, we used human embryonic stem cell (hESC)-derived CMs (hESC-CMs) as a tool to identify miRNAs that promotes CM proliferation. We profiled miRNA expression at early stage of CM differentiation and identified a list of highly expressed miRNAs. Among these miRNAs, miR-25 was enriched in early-stage hESC-CMs, but its expression decreased over time. Overexpression of miR-25 promoted CM proliferation. RNA-seq analysis revealed that genes related to cell cycle signal were strongly influenced by miR-25 overexpression. We further showed that miR-25 promoted CM proliferation by targeting FBXW7. Finally, the function of miR-25 in the regulation of CM proliferation was demonstrated in zebrafish. Our study suggested that miR-25 is a promising molecule for heart regeneration.

    更新日期:2020-01-21
  • Allele-selective knockdown of MYH7 using antisense oligonucleotides
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-21
    Brian R. Anderson; Marianne L. Jensen; Peter H. Hagedorn; Sean C. Little; Richard E. Olson; Ron Ammar; Bernadette Kienzle; John Thompson; Ivar McDonald; Stephen Mercer; Jonas Vikesaa; Bettina Nordbo; Larry Iben; Yang Cao; Joanne Natale; Greg Dalton-Kay; Angela Cacace; Bo R. Hansen; Linda J. Bristow

    Hundreds of dominant negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knockdown mutant myosin transcripts by targeting single nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

    更新日期:2020-01-21
  • Mbd2 mediates retinal cell apoptosis by targeting the lncRNA Mbd2-AL1/miR-188-3p/Traf3 axis in ischemia/reperfusion injury
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-18
    Yanni Ge; Ran Zhang; Yuqing Feng; Huiling Li

    Recent studies reported that DNA methylation was involved in retinal cell death. Methyl-CpG binding domain protein 2 (Mbd2) is one of DNA methylation readers. Its role and mechanism of regulation remain unclear. The ischemia/reperfusion (I/R) model in mice primary culture retinal ganglion cells and Mbd2 knock-out (Mbd2-KO) mice were used in the current study. We demonstrated that Mbd2 mediates RGCs apoptosis caused by I/R injury. Mechanistically, the data suggested that Mbd2 upregulated Mbd2-associated long non-coding RNA 1 (Mbd2-AL1) via demethylation of its promoter. Furthermore, Mbd2-AL1 sponged miR-188-3p, thus preventing TNF receptor associated factor 3 (Traf3) downregulation and inducing RGCs apoptosis. This was further demonstrated by the fact that inhibition of miR-188-3p diminished the anti-apoptosis role of Mbd2-AL1 siRNA. Finally, it showed that the apoptosis of retinal cells was attenuated and the visual function was preserved in Mbd2-KO mice which was associated with the Mbd2-AL1/miR-188-3p/Traf3 axis. Our present study revealed the role of Mbd2 in RGCs apoptosis which may provide a novel therapeutic strategy for retinal ischemic diseases.

    更新日期:2020-01-21
  • LncRNA RMST Suppressed GBM Cells Mitophagy through Enhancing FUS SUMOylation
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-18
    Changhong Liu; Zixuan Peng; Peiyao Li; Haijuan Fu; Jianbo Feng; Yan Zhang; Tao Liu; Yang Liu; Qing Liu; Qiang Liu; Di Li; Minghua Wu

    Background Long non-coding RNAs (lncRNAs) play significant role in post-translational modifications of proteins, yet the importance of lncRNAs for sumoylation is unknown. Methods RMST expression in glioma tissues and normal brain tissues was measured by RT-qPCR and in situ hybridization.The functional roles of RMST in astrocytomas were demonstrated by a series of in vitro experiments. The potentialmechanisms of RMST for sumoylation was investigatedby RNA immunoprecipitation,RNA pull-down,western blotting and co-immunoprecipitation assays. Findings We first demonstrated the oncogenic activity of lncRNA RMST by inhibiting glioma cells mitophagy. We also first determined that RMST is an enhancer of FUS SUMOylation, especially boosting SUMO1 modification at K333. SUMOylation induced by RMST contributes to the interaction between FUS and hnRNPD and stabilized their expression and cells mitophagy. Importantly, LncRNA RMST could serve as a promising prognostic factor for glioma patients. Interpretation our results demonstrated a previously unknown function of lncRNAs worked as an enhancer in FUS SUMOylation, and RMST will be a significant guide for the development of medications targeting gliomas.

    更新日期:2020-01-21
  • Identification of the regulatory role of lncRNA SNHG16 in myasthenia gravis by constructing a competing endogenous RNA network
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-18
    Jianjian Wang; Yuze Cao; Xiaoyu Lu; Xiaolong Wang; Xiaotong Kong; Chunrui Bo; Shuang Li; Ming Bai; Yang Jiao; Hongyu Gao; Xiuhua Yao; Shangwei Ning; Lihua Wang; Huixue Zhang

    Myasthenia gravis (MG) is an autoimmune disorder resulting from antibodies against the proteins at the neuromuscular junction. Emerging evidence indicates that lncRNAs, acting as competing endogenous RNAs (ceRNAs), are involved in various diseases. However, the regulatory mechanisms of ceRNA underlying MG remain largely unknown. In this study, we constructed a lncRNA-mediated ceRNA network involved in MG using a multi-step computational strategy. Functional annotation analysis suggests that these lncRNAs may play crucial roles in the immunological mechanism underlying MG. Importantly, through manual literature-mining, we found that lncRNA SNHG16, acting as a ceRNA, plays important roles in the immune processes. Further experiments showed that SNHG16 expression was up-regulated in peripheral blood mononuclear cells (PBMCs) from MG patients compared to healthy controls. Luciferase reporter assays confirmed that SNHG16 is a target of the miRNA let-7c-5p. Subsequent experiments indicated that SNHG16 regulates the expression of the key MG gene IL-10 by sponging let-7c-5p in a ceRNA manner. Furthermore, functional assays showed that SNHG16 inhibits Jurkat cell apoptosis and promotes cell proliferation by sponging let-7c-5p. Our study will contribute to a deeper understanding of the regulatory mechanism of MG and will potentially provide new therapeutic targets for MG patients.

    更新日期:2020-01-21
  • Transplantation of human mesenchymal stem cells genome-edited with LEF1 improves cardio-protective effects in myocardial infarction
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-18
    Hyun-Min Cho; Kang-Hoon Lee; Yi-ming Shen; Tae-Jin Shin; Pan-Dong Ryu; Min-Cheol Choi; Kyung-Sun Kang; Je-Yoel Cho

    Stem cell-based therapy is one of the most attractive approaches to ischemic heart diseases such as myocardial infarction (MI). We evaluated the cardio-protective effects of the hUCB-MSCs stably expressing LEF1 (LEF1/hUCB-MSCs) in a rat model of MI. LEF1 overexpression in hUCB-MSCs promoted cell proliferation and anti-apoptotic effects in hypoxic conditions. For the application of its therapeutic effects in vivo, the LEF1 gene was introduced into an AAVS1 locus known as a safe harbor site on chromosome 19 by CRISPR/Cas9-mediated gene integration in hUCB-MSCs. Transplantation of LEF1/hUCB-MSCs onto the infarction region in the rat model significantly improved overall survival. The cardio-protective effect of LEF1/hUCB-MSCs was proved by echocardiogram parameters including greatly improved left ventricle ejection fraction (EF) and fractional shortening (FS). Moreover, histology and immunohistochemistry successfully presented reduced MI region and fibrosis by LEF1/hUCB-MSCs. We found that this overall positive effects of LEF1/hUCB-MSCs are attributed by increased proliferation and survival of stem cells in oxidative stress conditions and by the secretion of various growth factors by LEF1. In conclusion, this study suggests that the stem cell-based therapy conjugated with genome editing of transcription factor LEF1, which promotes cell survival, could be an effective therapeutic strategy for cardiovascular disease.

    更新日期:2020-01-21
  • Long non-coding RNA NEAT1 binds to microRNA-339-5p to increase HOXA1 and alleviate ischemic brain damage in neonatal mice
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-17
    Jing Zhao; Ling He; Lingling Yin

    Hypoxic-ischemic brain damage (HIBD) is a major cause of fatality and morbidity in neonates. However, current treatment approaches to alleviate HIBD are not effective. Various studies have highlighted the role of microRNAs (miRNAs) in various biological functions in multiple diseases. This study investigated the role of miR-339-5p in HIBD progression. Neonatal HIBD mouse model was induced by ligation of the right common carotid artery. Neuronal cell model exposed to oxygen-glucose deprivation (OGD) was also established. The miR-339-5p expression in mouse brain tissues and neuronal cells was quantified and the effects of miR-339-5p on neuronal cell activity and apoptosis induced by hypoxia-ischemia were explored. The overexpression or knockdown of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in hippocampal neurons was used to determine the effect of lncRNA NEAT1 on the expression of miR-339-5p and homeobox A1 (HOXA1) and apoptosis. Short hairpin RNA targeting lncRNA NEAT1 and miR-339-5p antagomir were used in neonatal HIBD mice to identify their roles in HIBD. Our results revealed that miR-339-5p was downregulated in neonatal HIBD mice and neuronal cells exposed to OGD. Downregulated miR-339-5p promoted neuronal cell viability and suppressed apoptosis during hypoxia-ischemia. Moreover, lncRNA NEAT1 competitively bind to miR-339-5p to increase HOXA1 expression and inhibited neuronal cell apoptosis under hypoxic-ischemic conditions. The key observations of the current study present evidence demonstrating that lncRNA NEAT1 upregulated HOXA1 to alleviate HIBD in mice by binding to miR-339-5p.

    更新日期:2020-01-21
  • MiRNA-31 improves cognition and abolishes amyloid-β pathology by targeting APP and BACE1 in an animal model of Alzheimer’s disease
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-17
    Ana Teresa Barros-Viegas; Vítor Carmona; Elisabete Ferreiro; Joana Guedes; Pedro Cunha; Luís Pereira de Almeida; Catarina Resende de Oliveira; João Pedro de Magalhães; João Peça; Ana Luísa Cardoso
    更新日期:2020-01-21
  • LncRNA CCAT1 acts as a microRNA-218 sponge to increase gefitinib resistance in NSCLC by targeting HOXA1
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-17
    Xiang Jin; Xiuhua Liu; Zhen Zhang; Yinghui Guan

    Long non-coding RNA (lncRNA) colon cancer associated transcript-1 (CCAT1) has been reported to play important roles in the development and progression of multiple human malignancies. However, the functional role and molecular mechanism of CCAT1 on gefitinib resistance in non-small-cell lung cancer (NSCLC) are largely unclear. The aim of this study is to explore the roles of CCAT1 on gefitinib resistance in NSCLC and explore the underlying mechanisms. The qRT-PCR analysis was to investigate the expression pattern of CCAT1 in gefitinib resistant NSCLC patient tissues and cell lines. Then, the effects of CCAT1 on gefitinib resistance of NSCLC in vitro and in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of CCAT1 and miR-218 in NSCLC cells. In this study, CCAT1 was observed to be upregulated in gefitinib resistant patient tissues and cell lines. In vitro and in vivo experiments demonstrated that CCAT1 knockdown impaired cell proliferation and promoted the gefitinib-induced cell apoptosis. Furthermore, we demonstrated that CCAT1 acts as a sponge for miR-218 and verified that HOXA1 is a novel target of miR-218. These results suggest that CCAT1 may serve as a promising therapeutic target for the treatment of EGFR+ NSCLC patients.

    更新日期:2020-01-21
  • Strong immune responses induced by direct local injections of modified mRNA-lipid nanocomplex
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-16
    Smriti Arya; Qiubin Lin; Nan Zhou; Xiang Gao; Jian-Dong Huang

    In vitro transcribed mRNAs hold the promises of many medical applications in disease prevention and treatment, such as replacement or supplement of missing or inadequately expressed endogenous proteins and as preventive vaccines against infectious diseases, therapeutic vaccines or other protein-based biopharmaceutics for cancer therapy. A safe and efficient delivery system for mRNA is crucial to the success of mRNA therapeutic applications. In this study, we report that InstantFECT, a liposome-based transfection reagent, can pack pseudouridine incorporated mRNA into nanocomplex that are highly efficient in mediating in vivo transfection in multiple organs after local delivery. High levels of expression of EGFP and luciferase reporters after intra-tumoral and intramuscular injections were observed, which lasted for up to 96 hrs. Immunogenicity of antigens encoded by mRNA, delivered with nanocomplex was investigated by subcutaneous delivery of modified mRNAs encoding Staphylococcus aureus adenosine synthase A (AdsA) and a model tumor-associated antigen ovalbumin (OVA). Strong T cell responses were provoked by both mRNAs delivered. Therapeutic and protective treatment with OVA mRNA-liposome nanocomplex significantly inhibited B16-OVA tumor progression and increased mice survival. There was no sign of obvious toxicity related to the treatment both in tissue culture and in mice. An intravenous injection of the same dosage of the modified mRNA-lipid nanocomplex showed minimal transfection in major organs, indicating an excellent safety feature as the gene transfer occurred only at the injection sites, whereas i.v. injection with the same amount of mRNA complexed with a commercial transfection reagent Trans-IT showed luciferase expression in the spleen. In summary, InstantFECT cationic liposomes provide a safe and efficient in vivo locoregional delivery of mRNA and could be a useful tool for basic research and for the development of mRNA-based therapies.

    更新日期:2020-01-21
  • Upregulation of microRNA-101a suppresses chronic renal fibrosis by regulating histone demethylases KDM3A via blockade of the YAP-TGFβ-Smad signaling pathway
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-16
    Hong Ding; Yanyan Xu; Nan Jiang

    Renal fibrosis denotes a common complication of diabetic nephropathy and is a predominant cause of end-stage renal disease. Despite the association between microRNAs (miRNAs or miRs) and renal fibrosis, miRNAs have been reported to play a vital role in the development of chronic renal fibrosis. Therefore, the aim of the present study was to investigate the possible function of miR-101a in chronic renal fibrosis. Initially, microarray-based gene expression profiling of renal fibrosis was employed to screen the differentially expressed genes. An in vivo mouse model of chronic renal fibrosis induced by a unilateral ureteral obstruction (UUO) and an in vitro cell model induced by aristolochic acid (AA) were constructed. miR-101a expression was examined using FISH assay and RT-qPCR. Then, the interaction between miR-101a and KDM3A was identified using an online website combined with dual-luciferase reporter assay. Finally, gain- and loss-of-function experiments were conducted to elucidate the effect of miR-101a on the expression of Col1a1, fibronectin, α-SMA, and YAP-TGFβ-Smad signaling pathway-related genes, as well as the degree of renal fibrosis. miR-101a was poorly expressed while KDM3A was robustly induced in chronic renal fibrosis tissues and cells. In addition, miR-101a could target and downregulate KDM3A expression, which led to elevated TGIF1, inhibited expression of Col1a1, fibronectin, α-SMA, YAP1, and TGFβ2 along with the extent of Smad2/3 phosphorylation, as well as delayed renal fibrosis degree. Besides, overexpressed YAP/TGFβ2 or inhibited TGIF1 partially restored the inhibitory effect of miR-101a on chronic renal fibrosis. Taken together, miR-101a could potentially slow down chronic renal fibrosis by the inactivation of the YAP-TGFβ-Smad signaling pathway via KDM3A, highlighting the potential of miR-101a as a therapeutic target for chronic renal fibrosis treatment.

    更新日期:2020-01-21
  • Novel Mouse MicroRNA Chr13_novelMiR7354-5p Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-16
    Feng Zhao; Xiaoyu Liu; Zhe Wang; Hongxin Lang; Tao Zhang; Rui Wang; Xuewen Lin; Dan He; Ping Shi; Xining Pang

    MicroRNAs (miRNAs) that play key roles in generation of insulin-producing cells from stem cells provide a cell-based approach for insulin replacement therapy. Here, we used next-generation sequencing to detect the miRNA expression profile of normal mouse pancreatic β-cells, non-β-cells, bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived stem cells (ADSCs) and determined relative miRNA expression levels in mouse pancreatic β-cells. After the novel mouse miRNA candidates were identified using miRDeep 2.0, we found that Chr13_novelMiR7354-5p, a novel miRNA candidate, significantly promoted the differentiation of BM-MSCs into insulin-producing cells in vitro. Furthermore, Chr13_novelMiR7354-5p-transfected BM-MSCs reversed hyperglycaemia in STZ-treated diabetic mice. In addition, bioinformatics analyses, a luciferase reporter assay and Western blotting demonstrated that Chr13_novelMiR7354-5p targeted Notch1 and Rbpj. Our results provide compelling evidence of the existence of 65 novel mouse miRNA candidates and present a new treatment strategy to generate insulin-producing cells from stem cells.

    更新日期:2020-01-21
  • Development of a facile approach for generating chemically-modified CRISPR/Cas9 RNA
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-16
    Tristan Scott; Citradewi Soemardy; Kevin V. Morris

    The RNA-guided, modified type II prokaryotic clustered regularly interspaced palindromic repeats with CRISPR-associated proteins (CRISPR/Cas9) system represents a simple gene editing platform with applications in biotechnology and also potentially as a therapeutic modality. The system requires a small guide RNA (sgRNA) and a catalytic Cas9 protein to induced non-homologous end joining (NHEJ) at break sites resulting in the formation of inactivating mutations, or through homology-directed repair (HDR) can engineer in specific sequence changes. Although CRISPR/Cas9 is a powerful technology, the effects can be limited as a result of nuclease-mediated degradation of the RNA components. Significant research has focused on the solid-phase synthesis of CRISPR RNA components with chemically modified bases, but this approach is technically challenging and expensive. Development of a simple, generic approach to generate chemically modified CRISPR RNAs may broaden applications that require nuclease-resistant CRISPR components. We report here the development of a novel, functional U-replaced tracrRNA that can be in vitro transcribed with chemically stabilizing 2'F-pyrimidines. These data represent a unique and facile approach to generating chemically stabilized CRISPR RNA.

    更新日期:2020-01-21
  • CircRNA-AKT1 sequesters miR-942-5p to upregulate AKT1 and promote cervical cancer progression
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-16
    Rongying Ou; Laiming Mo; Huijing Tang; Shaolong Leng; Haiyan Zhu; Liang Zhao; Yi Ren; Yunsheng Xu

    Statistics show that the prognosis of cervical cancer (CC) is poor and the death rate of CC in advanced stage has been rising in recent years. Increasing evidences have demonstrated that circular RNAs serve as promising biomarkers in human cancers, including CC. Present study planned to find out the circRNA involved in CC and to explore its regulatory mechanism in CC. We discovered the new circRNA, circ-0033550, upregulated in CC. Its associated gene was AKT (also known as protein kinase B) serine/threonine kinase 1 (AKT1), so we renamed circ-0033550 as circ-AKT1. We confirmed the high expression of circ-AKT1 in CC samples and cell lines as well as circle structure of circ-AKT1. Functionally, gain- and loss-of-function experiments indicated that circ-AKT1 and AKT1 promoted CC cell proliferation and invasion. Moreover, circ-AKT1 and AKT1 were induced by TGF-β, and facilitated EMT (epithelial-mesenchymal transition) in CC. Mechanically, we illustrated that circ-AKT1 upregulated AKT1 by sponging miR-942-5p. Rescue assays confirmed the role of circ-AKT1/miR-942-5p/AKT1 axis in CC progression. In vivo assays validated that circ-AKT1 promoted tumor growth in CC. Overall, circRNA-AKT1 sequestered miR-942-5p to upregulate AKT1 and promote CC progression, which may offer a new molecular target for the treatment improvement of CC.

    更新日期:2020-01-21
  • LINC00167 regulates RPE differentiation by targeting the miR-203a-3p/SOCS3 axis
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-15
    Xue Chen; Ruxu Sun; Daidi Yang; Chao Jiang; Qinghuai Liu

    Increasing evidences have indicated that long non-coding RNAs (lncRNAs) play significant roles in various diseases; however, their roles in age-related macular degeneration (AMD) remain unclear. Dedifferentiation and dysfunction of retinal pigment epithelium (RPE) cells have been shown to contribute to AMD etiology by several studies. Herein, we found that lncRNA LINC00167 was down-regulated in RPE-choroid samples of AMD patients and dysfunctional RPE cells, and was consistently up-regulated along with RPE differentiation. In vitro study indicated that reduced endogenous LINC00167 expression resulted in RPE dedifferentiation, which was typified by attenuated expression of RPE markers, reduced vascular endothelial growth factor A secretion, accumulation of mitochondrial reactive oxygen species, and interrupted phagocytic ability. Mechanistically, LINC00167 functioned as a sponge for microRNA miR-203a-3p to restore the expression of the suppressor of cytokine signaling (SOCS3), which further inhibited the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Taken together, our study demonstrated that LINC00167 showed a protective role in AMD by maintaining RPE differentiation through LINC00167/miR-203a-3p/SOCS3 axis and might be a potential therapeutic target for AMD.

    更新日期:2020-01-15
  • 更新日期:2020-01-15
  • Upregulation of long noncoding RNA LINC00460 facilitates gastric cancer progression through epigenetically silencing CCNG2 by EZH2/LSD1 and indicates poor outcomes
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-15
    Jiebin Yang; Yikai Lian; Renzhi Yang; Yifan Lian; Jingtong Wu; Jingjing Liu; Keming Wang; Hongzhi Xu

    Non-protein-coding functional elements in the human genome in postgenomic biology field have been drawing great attention in recent years. Thousands of long noncoding RNAs (lncRNAs) have been found to be expressed in various tumors. Yet only a small proportion of these lncRNAs have been well characterized. We have demonstrated that LINC00460 could affect cell proliferation through epigenetic regulation of KLF2 and CUL4A in human colorectal cancer. However, the clinical significance and biological role of LINC00460 in gastric cancer (GC) remain largely unknown. In this research, we discovered that LINC00460 is remarkably up-regulated in GC tissues compared to the non-tumor tissues. Besides, LINC00460 served as an independent prognostic marker in GC. Functionally, proliferation of GC cells could be regulated by LINC00460 both in vitro and in vivo. RNA-seq analysis for the whole transcriptome indicated that LINC00460 may serve as a key regulatory factor in the tumorigenesis of GC. What’s more, the biological function of LINC00460 was mediated, to certain extent, by the direct interaction with enhancer of zeste homolog 2 (EZH2) and lysine (K)-specific demethylase 1A (LSD1) proteins. Further analyses indicated that LINC00460 promoted GC proliferation at least partly through the down-regulation of tumor suppressor-gene Cyclin G2 (CCNG2) which is mediated by EZH2 and LSD1. In conclusion, our results suggested that LINC00460 acted as an oncogene in GC to inhibit the expression of CCNG2 at least partly by binding with EZH2 and LSD1. Our study could provide additional insights into the development of novel target therapeutic methods for GC.

    更新日期:2020-01-15
  • Novel engineered programmable systems for ADAR-mediated RNA editing
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-15
    Guillermo Aquino-Jarquin

    One of the most prevalent forms of post-transcriptional RNA modification is the conversion of adenosine nucleosides to inosine (A-to-I), mediated by adenosine deaminase acting on RNA (ADAR) enzymes. The advent of the CRISPR/Cas systems inspires researchers to work actively in the engineering of programmable RNA-guided machines for basic research and biomedical applications. In this regard, CIRTS, RESCUE, RESTORE, and LEAPER are innovative RNA base editing platforms that have recently been engineered to perform programmable base conversions on target RNAs mediated by ADAR enzymes, in mammalian cells. Thus, these four currently characterized RNA editing systems constitute novel molecular tools with compelling programmability, specificity, and efficiency that show us some creative ways to take advantage of the engineered deaminases for precise base editing. Moreover, the advanced engineering of these systems permits to edit full-length transcripts containing disease-causing point mutations without the loss of genomic information, providing an attractive alternative for in-vivo research and in the therapeutic setting if the challenges encountered in off-target edits and delivery are appropriately addressed. Here, I present an analytical approach of the current status and rapid progress of the novel ADAR-mediated RNA editing systems when highlighting the qualities of each new RNA editing platform and how these RNA targeting strategies could be used to recruit human ADARs on endogenous transcripts, not only for our understanding of RNA-modification-mediated regulation of gene expression but also for editing clinically relevant mutations in a programmable and straightforward manner.

    更新日期:2020-01-15
  • miR-552 Regulates Liver Tumor-Initiating Cells Expansion and Sorafenib Resistance
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-15
    Tao Han; Yue Zhang; Xiaodan Yang; Lei Han; Hengyu Li; Tingsong Chen; Zhendong Zheng

    MicroRNAs (miRNAs) are involved in tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). However, few miRNAs have been identified and entered clinical practice. Herein, we report that miR-552 is upregulated in HCC tissues and has an important function in liver tumor-initiating cells (T-ICs). Functional studies revealed that a forced expression of miR-552 promotes liver T-ICs self-renewal and tumorigenesis. Conversely, miR-552 knockdown inhibits liver T-ICs self-renewal and tumorigenesis. Mechanistically, miR-552 downregulates PTEN via its mRNA 3’UTR and activates AKT phosphorylation. Our clinical investigations elucidated the prognostic value of miR-552 in HCC patients. Furthermore, miR-552 expression determines the responses of hepatoma cells to sorafenib treatment. The analysis of patient cohorts and patient-derived xenografts (PDXs) further demonstrated that miR-552 may predict sorafenib benefits in HCC patients. In conclusion, our findings revealed the crucial role of the miR-552 in liver T-ICs expansion and sorafenib response, rendering miR-552 an optimal target for the prevention and intervention in HCC.

    更新日期:2020-01-15
  • CircRASSF2 acts as competing endogenous RNA and promotes papillary thyroid carcinoma progression through miR-1178/TLR4 signaling pathway
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-15
    Wenhong Zhou; Guojun Wu; Jiyu Li; Xiaona Lin; Yongjie Sun; Hao Xu; Peng Shi; Ling Gao; Xingsong Tian

    Circular RNAs (circRNAs) are a class of non-coding RNAs broadly expressed in cells of various species. However, the molecular mechanisms that link circRNAs with progression of papillary thyroid carcinoma (PTC) are not well understood. In the present study, we attempted to provide novel basis for targeted therapy for PTC from the aspect of circRNA-miRNA-mRNA interaction. We investigated the expression of circRNAs in 5 paired PTC tissues and normal tissues by microarray analysis. The circRNA microarray assay followed by RT-qPCR was used to verify the differential expression of hsa_circ_0059354, which is located on chromosome 20 and derived from RASSF2, and thus we named it circRASSF2. The qRT-PCR analysis was to investigate the expression pattern of circRASSF2 in PTC tissues and cell lines. Then, the effects of circRASSF2 on cell proliferation and apoptosis were assessed in PTC in vitro. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of circRASSF2 and miR-1178 in PTC cells. In this study, circRASSF2 was observed to be upregulated in PTC tissues and cell lines. Knockdown of circRASSF2 inhibited cell proliferation and promoted cell apoptosis in PTC cells. Bioinformatics analysis predicted that there is a circRASSF2/miR-1178/TLR4 axis in PTC. Dual-luciferase reporter system validated the direct interaction of circRASSF2, miR-1178, and TLR4. Furthermore, circRASSF2 facilitates PTC progression in vivo. Importantly, we demonstrated that circRASSF2 was upregulated in serum exosomes from PTC patients. In summary, our study demonstrated that circRASSF2 modulates PTC progression through miR-1178/TLR4 pathway. Our findings indicated that circRASSF2 may serve as a promising therapeutic target for the treatment of PTC patients.

    更新日期:2020-01-15
  • Nanoparticle delivery of anti-inflammatory locked nucleic acid oligonucleotides prevents airway inflammation in a house dust mite model of asthma
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Sabrina C. Ramelli; Brian S. Comer; Jared M. McLendon; Lydia L. Sandy; Andrew P. Ferretti; Robert Barrington; Jeff Sparks; Majed Matar; Jason Fewell; William T. Gerthoffer
    更新日期:2020-01-15
  • Comparative Analysis of Single-cell Transcriptome Identify Reprogramming Driver Factor for Efficiency Improvement
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Hanshuang Li; Mingmin Song; Wuritu Yang; Pengbo Cao; Lei Zheng; Yongchun Zuo

    Terminally differentiated somatic cells can be reprogrammed into a totipotent state through somatic cell nuclear transfer (SCNT). The incompletely reprogramming is the major reason for developmental arrest of SCNT embryos at early stages. In our studies, we found pathways for autophagy, endocytosis and apoptosis were incompletely activated in NT 2-cell arrest embryos, whereas extensively inhibited pathways for stem cell pluripotency maintenance, DNA repair, cell cycle and autophagy may result in NT 4-cell embryos arrest. As for NT normal embryos, a significant shift in expression of developmental TFs Id1, Pou6f1, Cited1 and Zscan4c were observed. Compared with pluripotent gene Ascl2 only activated in NT 2-cell, Nanog, Dppa2 and Sall4 had major expression waves both in normal development of NT 2-cell and 4-cell embryos. Additionally, Kdm4b/4d and Kdm5b had been confirmed as key markers in NT 2-cell and 4-cell embryos, respectively. Histone acetylase Kat8, Elp6, and Eid1 were co-activated in NT 2 and 4-cell to facilitate embryos normal development. Gadd45a as a key driver functions with Tet1 and Tet2 to improve the efficiency of NT reprogramming. Taken together, our findings provided important theoretical basis for elucidating the potential molecular mechanisms and identified reprogramming driver factor to improve the efficiency of SCNT reprogramming.

    更新日期:2020-01-15
  • The circular RNA circHUWE1 sponges the miR-29b-AKT3 axis to regulate myoblast development
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Binglin Yue; Jian Wang; Wenxiu Ru; Jiyao Wu; Xiukai Cao; Haiyan Yang; Yongzheng Huang; Xianyong Lan; Chuzhao Lei; Bizhi Huang; Hong Chen

    Myogenesis is controlled by a well-established transcriptional hierarchy that coordinates the activities of a set of muscle genes. Rencently, roles in myogenesis have been described for non-coding RNAs, including a role of circular RNA (circRNA) to regulate muscle gene expression. However, the functions of circRNA and the underlying mechanism by which circRNAs affect myogenesis remain poorly understood. In this study, we analyzed circRNA high-throughput sequencing results of bovine skeletal muscle samples, and constructed a circRNA–miRNA–mRNA network according to the competitive endogenous RNA (ceRNA) theory. The putative circHUWE1-miR-29b-AKT3 network was analyzed and its involvement in myogenesis was confirmed through a series of assays. To assess the potential function of this regulation, bovine myoblasts were infected with overexpression plasmids and siRNAs that target circHUWE1. Next, cell proliferation, apoptosis, and differentiation were analyzed using CCK8, EdU, flow cytometry, Western blotting, and RT-qPCR assays. The results suggest that circHUWE1 facilitates bovine myoblast proliferation and inhibits cell apoptosis and differentiation. Next, bioinformatics, dual luciferase reporter assay, and AGO2 RNA immunoprecipitation (RIP) approaches were used to verify the interaction between circHUWE1, miR-29b, and AKT3. Subsequently, we identified that circHUWE1 could directly interfere with the ability of miR-29b to relieve AKT3 suppression, which ultimately activates the AKT signaling pathway. These findings suggested a new regulatory pathway for bovine skeletal muscle development, and also expand our understanding of circRNA functions in mammals.

    更新日期:2020-01-15
  • CRISPR-Cas12a Possess Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Bin Li; Jingyue Yan; Youxi Zhang; Wenqing Li; Chunxi Zeng; Weiyu Zhao; Xucheng Hou; Chengxiang Zhang; Yizhou Dong

    CRISPR-Cas12a (CRISPR-Cpf1) was reported to have multiple types of cleavage activities. Without the assistance of CRISPR RNA (crRNA), we investigated DNase activity and substrate specificity of Cas12a orthologs in the presence of diverse divalent metal ions. Cas12a from different species are capable of degrading single-stranded DNA (ssDNA) and (or) double-stranded DNA (dsDNA), depending on the metal ions used. In spite of sharing high sequence similarity and functional domains among diverse Cas12a orthologs, only Acidaminococcus sp. Cas12a (AsCas12a) showed a predominant preference for cleaving ssDNA, whereas no detectable activity toward dsDNA substrate in the presence of magnesium (II) ions. In addition, we found that both AsCas12a and Francisella novicida Cas12a (FnCas12a) caused substantial dsDNA cleavage in the presence of manganese (II) ion. More importantly, the DNase activities can be inhibited by synthetic DNA oligonucleotides with phosphorothioate linkage modifications. Overall, ssDNase activity of the Cas12a orthologs uncovered a distinct approach for DNA cleavage compared to crRNA-guided double stranded DNA breaks, and provided insights into potential biological and therapeutic applications.

    更新日期:2020-01-15
  • The biomarker TCONS_00016233 drives septic AKI by targeting the miR-22-3p/AIFM1 signaling axis
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Pan Zhang; Lei Yi; Siyuan Qu; Jinzhong Dai; Xiaozhou Li; Bohao Liu; Huiling Li; Kai Ai; Peilin Zheng; Shuangfa Qiu; Yijian Li; Yinhuai Wang; Xudong Xiang; Xiangping Chai; Zheng Dong; Dongshan Zhang

    The prediction of mortality for septic acute kidney injury (AKI) has been assessed by a number of potential biomarkers, including long non-coding RNAs (lncRNAs). However, the validation of lncRNAs as biomarkers, particularly for the early stages of septic AKI, is still warranted. Our results indicate that the lncRNA TCONS_00016233 is upregulated in plasma of sepsis-associated non-AKI and AKI patients, but a higher cutoff threshold (9.5x105, copy number) provided a sensitivity of 71.9% and specificity of 89.6% for the detection of AKI. The plasma TCONS_00016233 was highly correlated with serum creatinine, TIMP-2, IGFBP7, IL-1β, TNFα, CRP, and urinary TCONS_00016233. LPS induced the expression of lncRNA TCONS_00016233 via TLR4/p38MAPK signal pathway in human renal tubular epithelial (HK-2) cells. Furthermore, TCONS_00016233 mediates the LPS induced HK-2 cells apoptosis and the expression of IL-1β and TNFα. Mechanistically, TCONS_00016233 acts as a ceRNA to prevent miR-22-3p-mediated down-regulation of apoptosis-inducing factor mitochondrion- associated 1(AIFM1). Finally, overexpression of TCONS_00016233 is capable of aggravating the LPS and CLP-induced septic AKI by targeting miR-22-3p/AIFM1 axis. Taken together, our data indicate that TCONS_00016233 may serve as an early diagnosis marker for the septic AKI, possibly acting as a novel therapeutic target for septic AKI.

    更新日期:2020-01-15
  • Binding and structural properties of DNA aptamers with VEGF-A-mimic activity
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Toru Yoshitomi; Misako Hayashi; Takumi Oguro; Keiko Kimura; Fumiya Wayama; Hitoshi Furusho; Keitaro Yoshimoto
    更新日期:2020-01-15
  • CircINSR promotes proliferation and reduces apoptosis of embryonic myoblasts by sponging miR-34a
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-14
    Xuemei Shen; Xiaoyan Zhang; Wenxiu Ru; Yongzhen Huang; Xianyong Lan; Chuzhao Lei; Hong Chen

    As a diverse and abundant class of endogenous RNAs, circular RNAs (circRNAs) participate in processes including cell proliferation and apoptosis. Nevertheless, few researchers have investigated the function of circRNAs in bovine muscle development. Based on existing sequencing data, we identified circINSR. The localization of circINSR in bovine myoblasts was investigated by fluorescence in situ hybridization. Molecular and biochemical assays were used to confirm the role of circINSR in myoblast proliferation and the cell cycle. Mitochondrial membrane potential and annexinⅤ-PE/7-AAD staining assays were performed to assess cell apoptosis. Additionally, interactions between circINSR, miR-34a, and target mRNAs were examined using bioinformatics, luciferase assay, and RNA immunoprecipitation. We found that circINSR was highly expressed in embryonic muscle tissue. Overexpression of circINSR significantly promoted proliferation and reduced apoptosis of embryonic myoblasts. Our data suggested that circINSR may act as a sponge of miR-34a and could function through de-repression of target genes in muscle cells. This study proposes that circINSR may function as a regulator of embryonic muscle development. circINSR regulates cells proliferation and apoptosis through miR-34a-modulated Bcl-2 and CyclinE2 expression.

    更新日期:2020-01-14
  • MicroRNA-122 exerts inhibitory effects on osteoblast proliferation and differentiation in osteoporosis by activating the PCP4-mediated JNK signaling pathway
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-13
    Yi-Chen Meng; Tao Lin; Heng Jiang; Jia Yin; Xiao Ma; Rui Gao; Xu-Hui Zhou

    Osteoporosis is characterized by the reduction of bone mineral density and deterioration of bone quality, which leads to high risk of fractures. Some microRNAs (miRNAs) have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass maintenance. We aimed to clarify whether microRNA-122 (miR-122) could regulate osteoblast differentiation in ovariectomized rats with osteoporosis. miR-122 was upregulated and purkinje cell protein 4 (PCP4) was downregulated in ovariectomized rats. PCP4 was identified as a target of miR-122 by dual-luciferase reporter gene assay. We transfected isolated osteoblasts from ovariectomized rats with miR-122 mimic or inhibitor, or PCP4 overexpression vectors. Proliferation and differentiation of osteoblasts were repressed by the overexpression of miR-122 but enhanced by overexpression of PCP4. miR-122 could induce the activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, while PCP4 blocked this pathway. Rescue experiments further demonstrated that the inhibiting effects of miR-122 on osteoblast differentiation could be compensated by activation of the PCP4 or inhibition of JNK signaling pathway. Collectively, our data imply that miR-122 inhibits osteoblast proliferation and differentiation in rats with osteoporosis, highlighting a novel therapeutic target for osteoporotic patients.

    更新日期:2020-01-14
  • Upregulation of OIP5-AS1 predicts poor prognosis and contributes to the thyroid cancer cell proliferation and migration through activation of Wnt/β-catenin signaling pathway
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-11
    Qiuli Li; Weichao Chen; Rongzhen Luo; Zhiyi Zhang; Ming Song; Wenkuan Chen; Zhongyuan Yang; Yuanzhong Yang; Zhuming Guo; Ankui Yang

    As a common malignancy, thyroid cancer mainly occurs in endocrine system. There have been accumulating studies on therapeutic methods of thyroid cancer, but its internal molecular mechanism is still not fully understood. LncRNA OIP5-AS1 was confirmed as an oncogene and related to poor prognosis in various cancers. Nevertheless, its role and underlying mechanism remain unclear in thyroid cancer. Here, we observed a significant upregulation of OIP5-AS1 in thyroid cancer tissues and cells and upregulated OIP5-AS1 was correlated with poor prognosis in thyroid cancer. Moreover, OIP5-AS1 knockdown resulted in the inhibited cell proliferation and migration, while overexpressed OIP5-AS1 exhibited the reverse function in thyroid cancer. Besides, OIP5-AS1 was found to positively regulate Wnt/β-catenin signaling pathway. Through mechanism exploration, OIP5-AS1 was discovered to activate Wnt/β-catenin signaling pathway via FXR1/YY1/CTNNB1 axis. Finally, rescue assays indicated that the inhibitive role of silenced OIP5-AS1 in thyroid cancer cell growth and Wnt/β-catenin signaling pathway could be rescued by overexpression of CTNNB1 or addition of LiCl. In conclusion, upregulation of OIP5-AS1 predicted unfavorable prognosis and enhanced thyroid cancer cell growth by activating Wnt/β-catenin signaling pathway.

    更新日期:2020-01-11
  • LncRNA SLC7A11-AS1 promotes chemoresistance by blocking SCFβ-TRCP-mediated degradation of NRF2 in pancreatic cancer
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-11
    Qingzhu Yang; Kai Li; Xuemei Huang; Chen Zhao; Yu Mei; Xinyuan Li; Lin Jiao; Huanjie Yang

    Drug resistance is the major obstacle of gemcitabine-based chemotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC). Many long non-coding RNAs (lncRNAs) are reported to play vital roles in cancer initiation and progression. Here, we report that lncRNA SLC7A11-AS1 is involved in gemcitabine resistance of PDAC. SLC7A11-AS1 is overexpressed in PDAC tissues and gemcitabine resistant cell lines. Knockdown of SLC7A11-AS1 weakens the PDAC stemness and potentiates the sensitivity of resistant PDAC cells towards gemcitabine in vitro and in vivo. SLC7A11-AS1 promotes chemoresistance through reducing intracellular reactive oxygen species (ROS) by stabilizing NRF2, the key regulator in antioxidant defense. Mechanically, SLC7A11-AS1 is co-localized with β-TRCP1 in the nucleus. The exon 3 of SLC7A11-AS1 interacts with the F-box motif of β-TRCP1, the critical domain that recruits β-TRCP1 to the SCFβ-TRCP E3 complex. This interaction prevents the consequent ubiquitination and proteasomal degradation of NRF2 in the nucleus. Our results demonstrate that the overexpression of SLC7A11-AS1 in gemcitabine resistant PDAC cells can scavenge ROS by blocking SCFβ-TRCP-mediated ubiquitination and degradation of NRF2, leading to low level of intracellular ROS which is required for the maintenance of cancer stemness. These findings suggest SLC7A11-AS1 as a therapeutic target to overcome gemcitabine resistance for PDAC treatment.

    更新日期:2020-01-11
  • MicroRNA134 of ventral hippocampus involves in cocaine extinction-induced anxiety-like and depression-like behaviors in mice
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Yuehan Li; Xue Lu; Jiaxun Nie; Panpan Hu; Feifei Ge; Ti-Fei Yuan; Xiaowei Guan

    We previously found that cocaine abuse could increase microRNA134 (miR134) levels in the hippocampus. Yet, the roles of the miR134 in cocaine-related abnormal psychiatric outcomes remain unknown. In this study, using cocaine-induced conditioned place preference (CPP) mice model, we found that mice exhibit the enhanced anxiety-like and depression-like behaviors during cocaine extinction (CE) period of CPP, accompanied by obviously increased miR134 levels and decreased levels of nineteen genes which are associated with synaptic plasticity, glia activity and neurochemical microenvironments, in the ventral hippocampus (vHP). Knockdown of miR134 in vHP in vivo reversed the changes in fifteen of nineteen potential gene targets of miR134, and rescued the abnormal anxiety-like and depression-like behavioral outcomes in CE mice. In parallel, knockdown of miR134 reversed CE-induced changes in dendritic spines and synaptic proteins, and increased the field EPSP of CA1 pyramidal neurons in the vHP of CE mice. In addition, knockdown of miR134 suppressed the CE-enhanced microglia activity, inflammatory, apoptotic and oxidative stress statuses by in the vHP. Taken together, miR134 may involve in cocaine-associated psychiatric problems, potentially via regulating the expressions of its gene targets that are related to synaptic plasticity and neurochemical microenvironments.

    更新日期:2020-01-11
  • The Potential Therapeutic Role of Exosomal MicroRNA-520b Derived from Normal Fibroblasts in Pancreatic Cancer
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Xiangliang Zhang; Hui Li; Tiantian Zhen; Yu Dong; Xiaojuan Pei; Huijuan Shi

    Pancreatic cancer (PC) remains a major health concern, with conventional cancer treatments exerting little influence on the disease course. MicroRNA-520b (miR-520b) functions as a tumor suppressor in several types of human cancers, while its anti-tumor property in the context of PC is still fundamental. The aim of this study is to identify the potential therapeutic role of miR-520b transferred by exosomes derived from normal fibroblasts (NFs) in PC progression. A gain-of-function study was performed to examine the roles of miR-520b in PC cell line SW1990, which suggested that miR-520b served as a tumor suppressor in PC. In order to confirm the role of exosomal miR-520b, exosomes were isolated from NF culture medium and co-cultured with SW1990 cells. During the co-culture experiments, we disrupted exosome secretion and up-regulated exosomal miR-520b. The in vitro co-culture studies revealed that miR-520b was transferred from NF-derived exosomes to PC cells and thereby suppressed PC cell proliferation, invasion, migration, and stimulated apoptosis. Furthermore, inhibited tumor growth and live metastasis upon elevated miR-520b in exosomes was observed in vivo. Conjointly, our study demonstrates that NF-derived exosomal miR-520b impedes the progression of PC, which contributes to a novel therapeutic role of exosomal miR-520b for treating PC.

    更新日期:2020-01-11
  • A small molecule-controlled Cas9 repressible system
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Youjun Wu; Lu Yang; Tammy Chang; Fouad Kandeel; Jiing-Kuan Yee

    CRISPR-Cas9 has been developed into a powerful molecular tool for genome engineering, and has revolutionized the field of biomedical research. Despite the tremendous potential of CRISPR-Cas9 in biomedical research, precise control of CRISPR-Cas9 over the dose and exposure time is important to expand its applications. In this study, we fused Cas9 with a peptide termed small molecule-assisted shut-off (SMASh) consisting of a protease domain and a degron domain derived from hepatitis C virus (HCV). The presence of SMASh allows tight control of the Cas9 stability via a clinically approved HCV protease inhibitor asunaprevir (ASV). We showed that the engineered Cas9 responded to ASV administration and rapidly degraded in a dose- and time-dependent manner. Cas9 degradation was reversible upon ASV removal that restored the gene editing activity. We also showed that limiting the level of Cas9 in cells increased the specificity of gene editing. The SMASh tag therefore provides an effective tool to control Cas9 stability, allowing an improvement in the accuracy, safety and versatility of the CRISPR-Cas9 system for genome editing and gene regulation studies.

    更新日期:2020-01-11
  • Circular RNA profiling reveals exosomal circ_0006156 as novel biomarker in papillary thyroid cancer
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Guojun Wu; Wenhong Zhou; Xiaohua Pan; Zhigang Sun; Yongjie Sun; Hao Xu; Peng Shi; Jiyu Li; Ling Gao; Xingsong Tian

    Circular RNAs (circRNAs) are a class of non-coding RNAs that are broadly expressed in various biological cells and function in regulating gene expression. However, the molecular mechanisms that link circRNAs with progression of papillary thyroid carcinoma (PTC) are not well understood. In the present study, the function of circ_0006156 (circFNDC3B) was investigated in human PTC cells. First, we detected the expression of circFNDC3B in PTC tissues and PTC cell lines by RT-PCR. A luciferase reporter assay and AGO2-RIP was used to confirm the relationship between circFNDC3B and miR-1178. PTC cells were stably transfected with siRNA against circFNDC3B and cell proliferation, migration and invasion were detected to evaluate the effect of circFNDC3B in PTC, while tumorigenesis was assayed in nude mice. In this study, circFNDC3B was observed to be upregulated in PTC tissues and cell lines. Knockdown of circFNDC3B inhibited cell proliferation and promoted cell apoptosis in PTC cells. Bioinformatics analysis predicted that there is a circFNDC3B/miR-1178/TLR4 axis in PTC. Dual-luciferase reporter system validated the direct interaction of circFNDC3B, miR-1178, and TLR4. Furthermore, circFNDC3B facilitates PTC progression in vivo. Importantly, we demonstrated that circFNDC3B was upregulated in serum exosomes from PTC patients. In summary, our study demonstrated that circFNDC3B modulates PTC progression through miR-1178/TLR4 pathway. Our findings indicated that circFNDC3B may serve as a promising therapeutic target for the treatment of PTC patients.

    更新日期:2020-01-11
  • mmu_circ_0000790 Involves in the Pulmonary Vascular Remodeling in Mice with Hypoxic Pulmonary Hypertension via the microRNA-374c-mediated FOXC1
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Lei Yang; Huan Liang; Xianguo Meng; Li Shen; Zhanjiang Guan; Bingchang Hei; Haitao Yu; Shanshan Qi

    Recently, the identification of several circular RNAs (circRNAs) as vital regulators of microRNAs (miRNAs) underlines the increasing complexity of non-coding RNAs (ncRNAs)-mediated regulatory networks. This study aimed to explore the effects of mmu_circ_0000790 on the biological behaviors of pulmonary artery smooth muscle cells (PASMCs) in hypoxic pulmonary hypertension (HPH). The HPH mouse model and hypoxia-induced PASMC model were initially established, and the expression of mmu_circ_0000790 in the pulmonary vascular tissues and hypoxic PASMCs was determined using RT-qPCR. A series of in vitro experiments such as dual luciferase assay, RNA pull-down and RIP assays were conducted to evaluate the interactions among mmu_circ_0000790, microRNA-374c (miR-374c), and forkhead transcription factor 1 (FOXC1). The potential physiological functions of mmu_circ_0000790, miR-374c and FOXC1 in hypoxic PASMCs were investigated through gain- and loss-of function approaches. Upregulated mmu_circ_0000790 was found in both the HPH-pulmonary vascular tissues and hypoxic PASMCs. Besides, mmu_circ_0000790 could competitively bind to miR-374c and consequently upregulate the target gene of miR-374c, FOXC1. It was also observed that mmu_circ_0000790 induced proliferation and inhibited apoptosis of hypoxic PASMCs, which further promoted the pulmonary vascular remodeling in mice with HPH. Therefore, we speculate that mmu_circ_0000790 may serve as prospective targets for the treatment of patients with HPH.

    更新日期:2020-01-11
  • An Integrated Network-based Discovery of Clinically Approved Formula Fufang-Biejia-Ruangan Pill for Repositioning to Treat Hepatocellular Carcinoma by Inhibiting PI3K/AKT/NF-κB Activation
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Yanqiong ZHANG; Xia MAO; Wenjia CHEN; Xiaodong GUO; Liangxiang YU; Funeng Jiang; Xiaoyue WANG; Weijie LI; Qiuyan GUO; Taixian LI; Na LIN

    Drug repositioning offers new clinical applications for existing drugs with shorter approval processes, less costs and risks than de novo experimental drug development. Fufang-Biejia-Ruangan Pill (FBRP) is the first clinically approved anti-fibrosis herbal formula in China. Whether FBRP could be used to treat hepatocellular carcinoma (HCC) remains unclear. Herein, a total of 161 FBRP candidate targets against HCC were identified according to the topological importance in "hepatic fibrosis-cirrhosis-cancer axis-related gene-FBRP putative target" network, and mostly enriched in PI3K/AKT/NF-κB signaling. Experimentally, FBRP inhibited the liver fibrosis and prevented the development of neoplastic lesions at the early stages of hepatocarcinogenesis in diethylnitrosamine-induced rat HCC model. FBRP inhibited tumor cell proliferation, induced tumor-specific cell death and suppressed tumor progression in HCC rats with preventing the activation of PI3K, AKT and IKΚB proteins, reducing the nuclear accumulation of NFΚB1 protein, and decreasing the downstream proteins' expression. Consistently, FBRP suppressed HCC cell proliferation and induced cell cycle arrest in vitro. Co-treatment of FBRP with PI3K inhibitor exhibited additive inhibitory effect on PI3K/AKT/NF-κB activation. Collectively, our data showed the potentials of FBRP in hepatic fibrosis microenvironment regulation and tumor prevention, suggesting that FBRP may be a promising candidate drug for reduction of fibrogenesis and prevention of HCC.

    更新日期:2020-01-11
  • Hsa_circ_001653 Implicates in the Development of Pancreatic Ductal Adenocarcinoma by Regulating microRNA-377-Mediated HOXC6 Axis
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Xiangliang Zhang; Hui Li; Tiantian Zhen; Yu Dong; Xiaojuan Pei; Huijuan Shi

    Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive pancreatic cancer with poor survival rate. Circular RNAs (circRNAs) signatures have been identified in some human cancers, there are little data concerning their presence in PDAC. We investigated the role of hsa_circ_001653, a newly identified circRNA, in the development of PDAC. Hsa-circ-001653 expression was measured in 83 paired normal and tumor tissues surgically resected from PDAC patients. Phenotypic changes of PDAC cells were evaluated by assays for cell viability, cell cycle, invasion, and apoptosis. Tube-like structure formation of human umbilical vein endothelial cells (HUVECs) was examined in the presence of PDAC cells. Cross-talk between hsa_circ_001653 and miR-377/HOXC6 was assessed using dual-luciferase reporter assay, Ago2 immunoprecipitation, and northern blot analysis. Nude mice were inoculated with human PDAC cells for in vivo analysis. Hsa_circ_001653 was an upregulated circRNA in PDAC. Silencing of hsa_circ_001653 in PDAC cells via RNA interference inhibited cell viability, cell-cycle progression, in vitro angiogenesis, and invasive properties, showing pro-apoptotic effect. Hsa_circ_001653 was found to bind to miR-377, which in turn repressed HOXC6 expression. Inhibition of miR-377 by its specific inhibitor restored cell viability, cell-cycle progression, in vitro angiogenesis, and invasive properties in PDAC cells lacking endogenous hsa_circ_001653. When nude mice were inoculated with human PDAC cells, inhibition of hsa_circ_001653 had a therapeutic effect. Collectively, the present study provides an enhanced understanding of hsa_circ_001653 as a therapeutic target for PDAC.

    更新日期:2020-01-11
  • Upregulation of Circular RNA circATRNL1 to Sensitize Oral Squamous Cell Carcinoma to Irradiation
    Mol. Ther. Nucl. Acids (IF 5.919) Pub Date : 2020-01-10
    Guanhui Chen; Yiming Li; Yi He; Binghui Zeng; Chen Yi; Chao Wang; Xiliu Zhang; Wei Zhao; Dongsheng Yu

    Accumulating evidence has demonstrated that circular RNAs (circRNAs) play important roles in regulating gene expression involved in tumor development. However, the role of circRNAs in modulating the radiosensitivity of oral squamous cell carcinoma (OSCC) and its potential mechanisms have not been documented. We performed high-throughput RNA sequencing (RNA-seq) to investigate the circRNA expression profile in OSCC patients and discovered that the circATRNL1 expression was significantly downregulated and closely related to tumor progression. The circATRNL1 was structurally validated via Sanger sequencing, RNase R treatment, and specific convergent and divergent primer amplification. Importantly, the expression levels of circATRNL1 decreased after irradiation treatment, and upregulation of circATRNL1 enhanced the radiosensitivity of OSCC through suppressing proliferation and the colony survival fraction, inducing apoptosis and cell-cycle arrest. Moreover, we observed that circATRNL1 could directly bind to miR-23a-3p and relieve inhibition for the target gene PTEN. In addition, the tumor radiosensitivity-promoting effect of circATRNL1 overexpression was blocked by miR-23a-3p in OSCC. Further experiments also showed that PTEN can reverse the inhibitory effect of OSCC radiosensitivity triggered by miR-23a-3p. We concluded that circANTRL1 may function as the sponge of miR-23a-3p to promote PTEN expression and eventually contributes to OSCC radiosensitivity enhancement. This study indicates that circANTRL1 may be a novel therapeutic target to improve the efficiency of radiotherapy in OSCC.

    更新日期:2020-01-11
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