MicroRNA Modulation Induced by AICA Ribonucleotide in J1 Mouse ES Cells

ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs) are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells). It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells). AICA ribonucleotide (AICAR) is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for AICAR in ES cells pluripotency maintenance and give insight for its usage in iPS cells generation.     

LncRNA‐MIF,a c‐Myc‐activated long non‐coding RNA,suppresses glycolysis by promoting Fbxw7‐mediated c‐Myc degradation

The c‐Myc proto‐oncogene is activated in more than half of all human cancers. However, the precise regulation of c‐Myc protein stability is unknown. Here, we show that the lncRNA‐MIF (c‐Myc inhibitory factor), a c‐Myc‐induced long non‐coding RNA, is a competing endogenous RNA for miR‐586 and attenuates the inhibitory effect of miR‐586 on Fbxw7, an E3 ligase for c‐Myc, leading to increased Fbxw7 expression and subsequent c‐Myc degradation. Our data reveal the existence of a feedback loop between c‐Myc and lncRNA‐MIF, through which c‐Myc protein stability is finely controlled. Additionally, we show that the lncRNA‐MIF inhibits aerobic glycolysis and tumorigenesis by suppressing c‐Myc and miR‐586.     

MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

miR‐10a restores human mesenchymal stem cell differentiation by repressing KLF4

miRNAs have recently been shown to play a significant role in human aging. However, data demonstrating the effects of aging‐related miRNAs in human mesenchymal stem cells (hMSCs) are limited. We observed that hMSC differentiation decreased with aging. We also identified that miR‐10a expression was significantly decreased with age by comparing the miRNA expression of hMSCs derived from young and aged individuals. Therefore, we hypothesized that the downregulation of miR‐10a may be associated with the decreased differentiation capability of hMSCs from aged individuals. Lentiviral constructs were used to up‐ or downregulate miR‐10a in young and old hMSCs. Upregulation of miR‐10a resulted in increased differentiation to adipogenic, osteogenic, and chondrogenic lineages and in reduced cell senescence. Conversely, downregulation of miR‐10a resulted in decreased cell differentiation and increased cell senescence. A chimeric luciferase reporter system was generated, tagged with the full‐length 3′‐UTR region of KLF4 harboring the seed‐matched sequence with or without four nucleotide mutations. These constructs were cotransfected with the miR‐10a mimic into cells. The luciferase activity was significantly repressed by the miR‐10a mimic, proving the direct binding of miR‐10a to the 3′‐UTR of KLF4. Direct suppression of KLF4 in aged hMSCs increased cell differentiation and decreased cell senescence. In conclusion, miR‐10a restores the differentiation capability of aged hMSCs through repression of KLF4. Aging‐related miRNAs may have broad applications in the restoration of cell dysfunction caused by aging. J. Cell. Physiol. 228: 2324–2336, 2013. © The Authors. Published by Wiley Periodicals, Inc.     

miR379–410 cluster miRNAs regulate neurogenesis and neuronal migration by fine‐tuning N‐cadherin

N‐cadherin‐mediated adhesion is essential for maintaining the tissue architecture and stem cell niche in the developing neocortex. N‐cadherin expression level is precisely and dynamically controlled throughout development; however, the underlying regulatory mechanisms remain largely unknown. MicroRNAs (miRNAs) play an important role in the regulation of protein expression and subcellular localisation. In this study, we show that three miRNAs belonging to the miR379–410 cluster regulate N‐cadherin expression levels in neural stem cells and migrating neurons. The overexpression of these three miRNAs in radial glial cells repressed N‐cadherin expression and increased neural stem cell differentiation and neuronal migration. This phenotype was rescued when N‐cadherin was expressed from a miRNA‐insensitive construct. Transient abrogation of the miRNAs reduced stem cell differentiation and increased cell proliferation. The overexpression of these miRNAs specifically in newborn neurons delayed migration into the cortical plate, whereas the knockdown increased migration. Collectively, our results indicate a novel role for miRNAs of the miR379–410 cluster in the fine‐tuning of N‐cadherin expression level and in the regulation of neurogenesis and neuronal migration in the developing neocortex.     

MiR‐125b inhibits cell biological progression of Ewing's sarcoma by suppressing the PI3K/Akt signalling pathway

Objectives: Increasing evidence has suggested the close relationship between microRNAs (miRNAs) dysregulation and the carcinogenesis of Ewing's sarcoma (ES), among of which miR‐125b has been reported to be decreased in ES tissues recently. Strikingly, ectopic expression of miR‐125b could suppress cell proliferation of ES cell line A673, suggesting the tumor suppressor role of miR‐125b in ES. However, the other accurate mechanistic functions and relative molecule mechanisms are largely unknown. Materials and Methods: Herein, we completed a series of experiments to investigate the role of miR‐125b in Ewing's sarcoma. We restored the expression of miR‐125b in ES cell line A673 through transfection with miR‐125b mimics. To further understand the role of miR‐125b in ES, we detected the effects of miR‐125b on the cell proliferation, migration and invasion, cell cycle as well as cell apoptosis. Results: We found that restored expression of miR‐125b in ES cell line A673 inhibited cell proliferation, migration and invasion, arrested cell cycle progression, and induced cell apoptosis. Moreover, bioinformatic prediction suggested the oncogene, phosphoinositide‐3‐kinase catalytic subunit delta (PIK3CD), was a target gene of miR‐125b in ES cells. Further quantitative RT‐PCR and western blot assays identified over‐expression of miR‐125b suppressed the expression of PIK3CD mRNA and protein. PIK3CD participates in regulating the PI3K signaling pathway, which has been reported to play an important role in the development of ES. Suppression of PIK3CD down‐regulated the expression of phospho‐AKT and phospho‐mTOR proteins and inhibited the biologic progression of A673 cells. Conclusions: Collectively, these data suggest that miR‐125b functions as a tumor suppressor by targeting the PI3K/Akt/mTOR signaling pathway, and may provide potential therapy strategy for ES patients by targeting miRNA expression.     

MicroRNA‐488 inhibits endometrial glandular epithelial cell proliferation,migration, and invasion in endometriosis mice via Wnt by inhibiting FZD7

Endometriosis is a chronic inflammatory syndrome and nearly 6%‐10% of women are affected by it during the reproductive period. Previous studies have proved that microRNAs (miRNAs) are implicated in the pathogenesis of ovarian endometriosis. In this study, we aimed to investigate that restored miR‐488 would effectively inhibit the development of endometriosis. The microarray‐based data analysis was performed to screen endometriosis‐related differentially expressed genes (DEGs). The mouse model in endometriosis syndrome was established by being subcutaneously injected with Estradiol benzoate, and the ectopic endometrial tissues and normal endometrial tissues were collected. Additionally, the endometrial glandular epithelial cells were extracted from the endometrial glandular epithelial tissues from normal and endometriosis mice. In order to examine the role of miR‐488 in mice with endometriosis, we measured miR‐488 expression and expression levels of Frizzled‐7 (FZD7), cyclinD1, β‐catenin, and c‐Myc in vivo and in vitro. Finally, we detected the effect of miR‐488 on cell proliferation, apoptosis, migration and invasion in vitro. FZD7 was upregulated in human endometriosis. The data showed higher expression levels of FZD7, β‐catenin, c‐Myc and cyclinD1, and lower miR‐488 expression in mouse endometrial tissues. FZD7 was the target gene of miR‐488. Furthermore, elevated miR‐488 in isolated mouse endometrial glandular endometrial cells inhibited FZD7, the translocation of β‐catenin to nucleus, the activation of Wnt pathway, and the cell proliferation, migration and invasion. Collectively, these findings indicated that up‐regulated miR‐488 may reduce the proliferation, migration and invasion of endometrial glandular epithelial cells through inhibiting the activation of Wnt pathway by down‐regulating FZD7.     

Differential expression of basal microRNAs’ patterns in human dental pulp stem cells

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real‐time PCR. Notably, we observed 19 up‐regulated miRNAs and 29 significantly down‐regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM‐MSCs). The 19 up‐regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa‐miR‐516a‐3p, hsa‐miR‐125b‐1‐3p, hsa‐miR‐221‐5p, hsa‐miR‐7, hsa‐miR‐584‐5p, hsa‐miR‐190a, hsa‐miR‐106a‐5p, hsa‐mir‐376a‐5p, hsa‐mir‐377‐5p and hsa‐let‐7f‐2‐3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa‐miR‐516a‐3p and hsa‐miR‐7‐5p as these miRNAs were highly expressed upon validation with qRT‐PCR analysis. We further proceeded with loss‐of‐function analysis with these miRNAs and we observed that hsa‐miR‐516a‐3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa‐miR‐7‐5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa‐miR‐516a‐3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.     

MiR‐485‐3p modulates neural stem cell differentiation and proliferation via regulating TRIP6 expression

Recent references have showed crucial roles of several miRNAs in neural stem cell differentiation and proliferation. However, the expression and role of miR‐485‐3p remains unknown. In our reference, we indicated that miR‐485‐3p expression was down‐regulated during NSCs differentiation to neural and astrocytes cell. In addition, the TRIP6 expression was up‐regulated during NSCs differentiation to neural and astrocytes cell. We carried out the dual‐luciferase reporter and found that overexpression of miR‐485‐3p decreased the luciferase activity of pmirGLO‐TRIP6‐wt but not the pmirGLO‐TRIP6‐mut. Ectopic expression of miR‐485‐3p decreased the expression of TRIP6 in NSC. Ectopic miR‐485‐3p expression suppressed the cell growth of NSCs and inhibited nestin expression of NSCs. Moreover, elevated expression of miR‐485‐3p decreased the ki‐67 and cyclin D1 expression in NSCs. Furthermore, we indicated that miR‐485‐3p reduced proliferation and induced differentiation of NSCs via targeting TRIP6 expression. These data suggested that a crucial role of miR‐485‐3p in self‐proliferation and differentiation of NSCs. Thus, altering miR‐485‐3p and TRIP6 modulation may be one promising therapy for treating with neurodegenerative and neurogenesis diseases.     

MicroRNA‐539 functions as a tumour suppressor in prostate cancer via the TGF‐β/Smad4 signalling pathway by down‐regulating DLX1

Prostate cancer (PCa) is the second leading cause of cancer‐related death in males, primarily due to its metastatic potential. The present study aims to identify the expression of microRNA‐539 (miR‐539) in PCa and further investigate its functional relevance in PCa progression both in vitro and in vivo. Initially, microarray analysis was conducted to obtain the differentially expressed gene candidates and the regulatory miRNAs, after which the possible interaction between the two was determined. Next, ectopic expression and knock‐down of the levels of miR‐539 were performed in PCa cells to identify the functional role of miR‐539 in PCa pathogenesis, followed by the measurement of E‐cadherin, vimentin, Smad4, c‐Myc, Snail1 and SLUG expression, as well as proliferation, migration and invasion of PCa cells. Finally, tumour growth was evaluated in nude mice through in vivo experiments. The results found that miR‐539 was down‐regulated and DLX1 was up‐regulated in PCa tissues and cells. miR‐539 was also found to target and negatively regulate DLX1 expression, which resulted in the inhibition of the TGF‐β/Smad4 signalling pathway. Moreover, the up‐regulation of miR‐539 or DLX1 gene silencing led to the inhibition of PCa cell proliferation, migration, invasion, EMT and tumour growth, accompanied by increased E‐cadherin expression and decreased expression of vimentin, Smad4, c‐Myc, Snail1 and SLUG. In conclusion, the overexpression of miR‐539‐mediated DLX1 inhibition could potentially impede EMT, proliferation, migration and invasion of PCa cells through the blockade of the TGF‐β/Smad4 signalling pathway, highlighting a potential miR‐539/DLX1/TGF‐β/Smad4 regulatory axis in the treatment of PCa.