miR‐15b‐5p facilitates the tumorigenicity by targeting RECK and predicts tumour recurrence in prostate cancer

MicroRNAs (miRNAs) have been reported to participate in many biological behaviours of multiple malignancies. Recent studies have shown that miR‐15b‐5p (miR‐15b) exhibits dual roles by accelerating or blocking tumour progression. However, the molecular mechanisms by which miR‐15b contributes to prostate cancer (PCa) are still elusive. Here, miR‐15b expression was found significantly up‐regulated in PCa in comparison with the normal samples and was positively correlated with age and Gleason score in patients with PCa. Notably, PCa patients with miR‐15b high expression displayed a higher recurrence rate than those with miR‐15b low expression (P = 0.0058). Knockdown of miR‐15b suppressed cell growth and invasiveness in 22RV1 and PC3 cells, while overexpression of miR‐15b reversed these effects. Then, we validated that RECK acted as a direct target of miR‐15b by dual‐luciferase assay and revealed the negative correlation of RECK with miR‐15b expression in PCa tissues. Ectopic expression of RECK reduced cell proliferation and invasive potential and partially abrogated the tumour‐promoting effects caused by miR‐15b overexpression. Additionally, miR‐15b knockdown inhibited tumour growth activity in a mouse PCa xenograft model. Taken together, our findings indicate that miR‐15b promotes the progression of PCa cells by targeting RECK and represents a potential marker for patients with PCa.     

Micro‐RNAs meet epigenetics to make for better brains

Recent studies have highlighted the importance of regulatory non‐coding RNAs and epigenetics in controlling the differentiation of somatic stem cells. Two major pathways characterize these fields: micro‐RNAs (miRNAs) and DNA methylation. In this issue of EMBO Reports, Lv et al show that during mammalian corticogenesis, miR‐15b inhibits cytosine demethylation by targeting Tet3, a key methylcytosine dioxygenase. This leads to the epigenetic downregulation of cyclin D1. As a result, cell cycle and differentiation of neural progenitors are altered, promoting their switch to neurogenesis. Hence, Lv et al elegantly bring together miRNAs and DNA methylation in the cell cycle control of neural progenitors and neurogenesis.     

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits

Objective: MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein–Barr virus (EBV)‐transformed lymphoblastoid cell line (LCL) and associated clinical traits. Material and Methods: Hence, we performed miRNA microarray approach with early‐ (p6) and late‐passage (p161) LCLs. Results and Conclusion: Microarray data showed that nine miRNAs (miR‐20b*, miR‐28‐5p, miR‐99a, miR‐125b, miR‐151‐3p, miR‐151:9.1, miR‐216a, miR‐223* and miR‐1296) were differentially expressed in most LCLs during long‐term culture. In particular, miR‐125b was up‐regulated in all the tested late‐passage LCLs. miR‐99a, miR‐125b, miR‐216a and miR‐1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long‐term culture in a previous study. Linear regression analysis showed that miR‐200a and miR‐296‐3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor’s health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV‐mediated tumorigenesis.     

MiRNA‐145‐5p prevents differentiation of oligodendrocyte progenitor cells by regulating expression of myelin gene regulatory factor

The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR‐145‐5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR‐145‐5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG⁺ cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR‐145‐5p knockdown OPCs. Further, knockdown of miR‐145‐5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL‐specific genes targeted by miR‐145‐5p that exhibited upregulation with miR‐145‐5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR‐145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR‐145‐5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR‐145‐5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR‐145‐5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as‐yet‐unidentified mechanisms. These findings clarify the need for differential regulation of miR‐145‐5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR‐145‐5p is dysregulated.     

Epigenetic regulation of miR‐129‐2 and its effects on the proliferation and invasion in lung cancer cells

MicroRNAs (miRNAs) play a pivotal role in carcinogenesis. Dysregulation of miRNAs, both oncogenic miRNAs and tumour‐suppressive miRNAs, is closely associated with cancer development and progression. The levels of miRNAs could be changed epigenetically by DNA methylation in the 5′ untranslated region (UTR) of pre‐mature miRNAs. To investigate whether DNA methylation alters the expression of miR‐129 in lung cancer, we did DNA methylation assays and found that 5′ UTR region of miR‐129‐2 gene was absolutely methylated in both A549 and SPCA‐1 lung cancer cells, but totally un‐methylated in 95‐D cells. The expression of miR‐129 was restored by 5‐Aza‐2'‐deoxycytidine (DAC), a de‐methylation agent, in both A549 and SPCA‐1 cells, resulting in attenuated cell migration and invasion ability, and decreased protein level of NF‐κB, which indicates the involvement of NF‐κB pathway. To further illustrate the roles of miR‐129 in lung tumourigenesis, we overexpressed miR‐129 in lung cancer cells by transfection of miR‐129 mimics, and found arrested cell proliferation at G2/M phase of cell cycle and inhibited cell invasion. These findings strongly suggest that miR‐129 is a tumour suppressive miRNA, playing important roles in the development and progression of human lung cancer.     

MiR‐34b‐3p represses cell proliferation,cell cycle progression and cell apoptosis in non‐small‐cell lung cancer (NSCLC) by targeting CDK4

Lung cancer is the most common incident cancer, with a high mortality worldwide, and non‐small‐cell lung cancer (NSCLC) accounts for approximately 85% of cases. Numerous studies have shown that the aberrant expression of microRNAs (miRNAs) is associated with the development and progression of cancers. However, the clinical significance and biological roles of most miRNAs in NSCLC remain elusive. In this study, we identified a novel miRNA, miR‐34b‐3p, that suppressed NSCLC cell growth and investigated the underlying mechanism. miR‐34b‐3p was down‐regulated in both NSCLC tumour tissues and lung cancer cell lines (H1299 and A549). The overexpression of miR‐34b‐3p suppressed lung cancer cell (H1299 and A549) growth, including proliferation inhibition, cell cycle arrest and increased apoptosis. Furthermore, luciferase reporter assays confirmed that miR‐34b‐3p could bind to the cyclin‐dependent kinase 4 (CDK4) mRNA 3′‐untranslated region (3′‐UTR) to suppress the expression of CDK4 in NSCLC cells. H1299 and A549 cell proliferation inhibition is mediated by cell cycle arrest and apoptosis with CDK4 interference. Moreover, CDK4 overexpression effectively reversed miR‐34‐3p‐repressed NSCLC cell growth. In conclusion, our findings reveal that miR‐34b‐3p might function as a tumour suppressor in NSCLC by targeting CDK4 and that miR‐34b‐3p may, therefore, serve as a biomarker for the diagnosis and treatment of NSCLC.     

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‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR‐23a and miR‐374

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR‐23a and miR‐374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR‐23a during E10–19, PN1–7, and adult retina revealed an inverse relation with Brn3b expression. Though miR‐374 did not show such a pattern, its co‐expression with miR‐23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR‐23a and miR‐374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co‐ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1155–1171, 2014     

Differential expression of microRNAs in TM3 Leydig cells of mice treated with brain‐derived neurotrophic factor

Brain‐derived neurotrophic factor (BDNF) is a neurotrophin that can promote the development and proliferation of neurons. BDNF has been found to be involved in male reproduction. Leydig cells in testicular interstitial tissues can secrete testosterone in a luteinizing hormone‐dependent manner. We showed that BDNF and its receptor TrkB were expressed in mice TM3 Leydig cells in the present study. Furthermore, BDNF can promote proliferation of mouse TM3 Leydig cells in vitro. Results of microRNA (miRNA) deep sequencing showed that BDNF can alter the expression profile of miRNAs in TM3 Leydig cells. Eighty‐three miRNAs were significantly different in the BDNF‐treated and control groups (fold change of >2.0 or <0.5, P < 0.05) wherein 40 were upregulated and 43 were downregulated. The expression levels of miR‐125a‐5p, miR‐22‐5p, miR‐342‐59, miR‐451a, miR‐148a‐5p, miR‐29b‐3p, miR‐199b‐5p, and miR‐145a‐5p were further confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs participate in the pathways involved in signal transduction, cancer, metabolism, endocrine system, immune system, and nerve system. This study indicated that miRNAs might be involved in the BDNF‐regulated cellular functions of Leydig cells.     

MicroRNA‐194 protects against chronic hepatitis B‐related liver damage by promoting hepatocyte growth via ACVR2B

Persistent infection with the hepatitis B virus leads to liver cirrhosis and hepatocellular carcinoma. MicroRNAs (miRNAs) play an important role in a variety of biological processes; however, the role of miRNAs in chronic hepatitis B (CHB)‐induced liver damage remains poorly understood. Here, we investigated the role of miRNAs in CHB‐related liver damage. Microarray analysis of the expression of miRNAs in 22 CHB patients and 33 healthy individuals identified miR‐194 as one of six differentially expressed miRNAs. miR‐194 was up‐regulated in correlation with increased liver damage in the plasma or liver tissues of CHB patients. In mice subjected to 2/3 partial hepatectomy, miR‐194 was up‐regulated in liver tissues in correlation with hepatocyte growth and in parallel with the down‐regulation of the activin receptor ACVR2B. Overexpression of miR‐194 in human liver HL7702 cells down‐regulated ACVR2B mRNA and protein expression, promoted cell proliferation, acceleratedG1 to S cell cycle transition, and inhibited apoptosis, whereas knockdown of miR‐194 had the opposite effects. Luciferase reporter assays confirmed that ACVR2B is a direct target of miR‐194, and overexpression of ACVR2B significantly repressed cell proliferation and G1 to S phase transition and induced cell apoptosis. ACVR2B overexpression abolished the effect of miR‐194, indicating that miR‐194 promotes hepatocyte proliferation and inhibits apoptosis by down‐regulating ACVR2B. Taken together, these results indicate that miR‐194 plays a crucial role in hepatocyte proliferation and liver regeneration by targeting ACVR2B and may represent a novel therapeutic target for the treatment of CHB‐related liver damage.     

MicroRNA‐130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/β‐catenin pathway

More and more studies indicate the relevance of miRNAs in inducing certain drug resistance. Our study aimed to investigate whether microRNA‐130b‐3p (miR‐130b) mediates the chemoresistance as well as proliferation of lung cancer (LC) cells. MTS assay and apoptosis analysis were conducted to determine cell proliferation and apoptosis, respectively. Binding sites were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT‐PCR and immunoblot, respectively. Mouse xenograft model was used to evaluate the role of miR‐130b in cisplatin resistance in vivo. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR ) versus its parental cell lines, indicated its crucial relevance for LC biology. We identified PTEN as miR‐130b's major target and inversely correlated with miR‐130b expression in LC. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. Suppression of miR‐130b enhanced drug cytotoxicity and reduced proliferation of A549/CR cells both internally and externally. Particularly, miR‐130b mediated Wnt/β‐catenin signalling pathway activities, chemoresistance and proliferation in LC cell, which was partially blocked following knockdown of PTEN. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR) versus its parental cell lines, indicated its crucial relevance for LC biology. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway.     

MiR‐520b promotes the progression of non‐small cell lung cancer through activating Hedgehog pathway

Although the non‐small cell lung cancer (NSCLC) is one of the most malignant tumours worldwide, the mechanisms controlling NSCLC tumourigenesis remain unclear. Here, we find that the expression of miR‐520b is up‐regulated in NSCLC samples. Further studies have revealed that miR‐520b promotes the proliferation and metastasis of NSCLC cells. In addition, miR‐520b activates Hedgehog (Hh) pathway. Inhibitor of Hh pathway could relieve the oncogenic effect of miR‐520b upon NSCLC cells. Mechanistically, we demonstrate that miR‐520b directly targets SPOP 3′‐UTR and decreases SPOP expression, culminating in GLI2/3 stabilization and Hh pathway hyperactivation. Collectively, our findings unveil that miR‐520b promotes NSCLC tumourigenesis through SPOP‐GLI2/3 axis and provide miR‐520b as a potential diagnostic biomarker and therapeutic target for NSCLC.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.