MicroRNA‐760 Inhibits Doxorubicin Resistance in Hepatocellular Carcinoma through Regulating Notch1/Hes1‐PTEN/Akt Signaling Pathway

Accumulating studies have suggested that microRNA‐760 (miR‐760) plays an important role in chemoresistance of various cancer cells. However, whether miR‐760 regulates the chemoresistance of hepatocellular carcinoma (HCC) remains unclear. In this study, we found that miR‐760 was decreased in HCC cell lines, and doxorubicin (Dox) treatment significantly decreased miR‐760 expression in HCC cells. Overexpression of miR‐760 sensitized HCC cells to Dox‐induced cytotoxicity and apoptosis, whereas miR‐760 inhibition showed the opposite effects. Notch1 was predicted as a target gene of miR‐760. miR‐760 negatively regulated Notch1 expression and Notch1/Hes1 signaling. Overexpression of miR‐760 increased PTEN expression and decreased the phosphorylation of Akt. Activation of Notch signaling significantly reversed the inhibitory effect of miR‐760 on Dox‐resistance and abrogated the effect of miR‐760 on the PTEN/Akt signaling pathway in HCC cells. Overall, our results demonstrate that miR‐760 inhibits Dox‐resistance in HCC cells through inhibiting Notch1 and promoting PTEN expression.     

LncRNA NR2F1‐AS1 regulates hepatocellular carcinoma oxaliplatin resistance by targeting ABCC1 via miR‐363

Emerging evidence has validated the vital role of long non‐coding RNA (lncRNA) in the chemoresistance of cancer treatment. In the present study, we investigate the function of lncRNA NR2F1‐AS1 on oxaliplatin (OXA) resistance of hepatocellular carcinoma (HCC) and discover the underlying molecular mechanism. Results revealed that lncRNA NR2F1‐AS1 was up‐regulated in oxaliplatin‐resistant HCC tissue and cells using microarray analysis and RT‐PCR. Meanwhile, ABCC1 protein was overexpressed in OXA‐resistant HCC cells (Huh7/OXA and HepG2/OXA). In vitro, NR2F1‐AS1 knockdown reduced the invasion, migration, drug‐resistant gene (MDR1, MRP5, LRP1) and IC50 value in Huh7/OXA and HepG2/OXA cells. In vivo, NR2F1‐AS1 knockdown decreased the tumour weight of HCC cells. Bioinformatics tools and luciferase reporter assay confirmed miR‐363 targeted the 3′‐UTR of NR2F1‐AS1 and ABCC1 mRNA, presenting that NR2F1‐AS1 promoted ABCC1 expression through endogenous sponging miR‐363. In summary, results conclude that NR2F1‐AS1 regulates HCC OXA resistance through targeting miR‐363‐ABCC1 pathway, providing a vital theoretic mechanism and therapeutic target for HCC chemoresistance.     

lncRNA‐POIR promotes epithelial–mesenchymal transition and suppresses sorafenib sensitivity simultaneously in hepatocellular carcinoma by sponging miR‐182‐5p

Sorafenib (SOR) resistance remains a major obstacle in the effective treatment of hepatocellular carcinoma (HCC). A number of long noncoding RNAs (lncRNAs) are responsible for this chemoresistance. This study aimed to reveal the essential function of a recently defined lncRNA, lncRNA‐POIR, in the epithelial–mesenchymal transition (EMT) and SOR sensitivity of HCC cells. SOR‐induced cytotoxicity was analyzed via cell counting kit‐8 and ethynyl‐2'‐deoxyuridine incorporation assays, whereas immunoblotting and confocal immunofluorescence were used to determine the expression levels of EMT markers. Furthermore, loss‐ or gain‐of‐function approaches were used to demonstrate the role of lncRNA‐POIR/miR‐182‐5p on EMT and SOR sensitivity in HCC. The direct interaction between lncRNA‐POIR and miR‐182‐5p was verified using a luciferase reporter assay. We found that knockdown of lncRNA‐POIR sensitized HCC cells to SOR and simultaneously reversed EMT. As expected, miR‐182‐5p was confirmed as the downstream target of lncRNA‐POIR. Moreover, miR‐182‐5p overexpression clearly reversed EMT and promoted SOR‐induced cytotoxicity in representative HCC cells, whereas miR‐182‐5p downregulation played a contrasting role; miR‐182‐5p knockdown abolished the modulatory effects of lncRNA‐POIR siRNA on EMT and SOR sensitivity. Together, these pieces of data suggest that lncRNA‐POIR promotes EMT progression and suppresses SOR sensitivity simultaneously by sponging miR‐182‐5p. Thus, we proposed a compelling rationale for the use of lncRNA‐POIR as a promising predictor of SOR response and as a potential therapeutic target for HCC treatment in the future.     

MiR‐106b regulates the apoptosis and tumorigenesis of hepatocellular carcinoma via targeting Zinc finger and BTB domain‐containing protein 7A (Zbtb7a)

MicroRNAs play vital regulatory roles in various type of tumorigenesis. We aimed to explore the functional microRNAs that might play as therapeutic targets in hepatocellular carcinoma (HCC). In this study, our results revealed that microRNA‐106b was significantly increased in HCC tumor tissues. However, miR‐106b knockdown remarkably suppressed the growth and increased the apoptosis of Hub‐7 HCC cells. Biological analysis indicated that miR‐106b directly targeted toZinc finger and BTB domain‐containing protein 7A (Zbtb7a) to regulate the apoptosis of Hub‐7 cells. Extensively, Zbtb7a overexpression reversed Huh‐7 cell apoptosis and growth in vitro. Furthermore, in vivo studies confirmed that miR‐106b inhibition or Zbtb7a overexpression retarded the growth of Hub‐7 xenograft tumor in nude mice. In conclusion, we provide the evidence for the regulatory role of miR‐106b in HCC, which is causally linked to targeting of Zbtb7a. This study may provide miR‐106b as a potential therapeutic strategy for HCC.     

Up‐regulation of miR‐10b‐3p promotes the progression of hepatocellular carcinoma cells via targeting CMTM5

In this study, we investigated how miR‐10b‐3p regulated the proliferation, migration, invasion in hepatocellular carcinoma (HCC) at both in vitro and in vivo levels. CMTM5 was among the differentially expressed genes (data from TCGA). The expression of miR‐10b‐3p and CMTM5 was detected by qRT‐PCR and Western blot (WB). TargetScan was used to acquire the binding sites. Dual‐luciferase reporter gene assay was used to verify the direct target relationship between miR‐10b‐3p and CMTM5. WB analysis proved that miR‐10b‐3p suppressed CMTM5 expression. Furthermore, proliferation, invasion and migration of HCC cells were measured by MTT assay, colony formation assay, transwell assay and wound‐healing assay, respectively. Kaplan‐Meier plotter valued the overall survival of CMTM5. Finally, xenograft assay was also conducted to verify the effects of miR‐10b‐3p/CMTM5 axis in vivo. Up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were detected in HCC tissues and cell lines. CMTM5 was verified as a target gene of miR‐10b‐3p. The overexpression of CMTM5 contributed to the suppression of the proliferative, migratory and invasive abilities of HCC cells. Moreover, the up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were observed to be associated with worse overall survival. Lastly, we have confirmed the carcinogenesis‐related roles of miR‐10b‐3p and CMTM5 in vivo. We concluded that the up‐regulation of miR‐10b‐3p promoted the progression of HCC cells via targeting CMTM5.     

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‐129‐5p promotes docetaxel resistance in prostate cancer by down‐regulating CAMK2N1 expression

This study focuses on the effect of miR‐129‐5p on docetaxel‐resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT‐PCR in PCa patient tissues and cell lines including PC‐3 and PC‐3‐DR. Cells transfected with miR‐129‐5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR‐129‐5p and CAMK2N1 levels were identified by qRT‐PCR and dual‐luciferase reporter assay. CAMK2N1 was found to be down‐expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up‐regulation of miR‐129‐5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR‐129‐5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR‐129‐5p contributed to the resistance of PC‐3‐DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR‐129‐5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.     

Long noncoding RNA MALAT1 enhances the docetaxel resistance of prostate cancer cells via miR‐145‐5p‐mediated regulation of AKAP12

Our present work was aimed to study on the regulatory role of MALAT1/miR‐145‐5p/AKAP12 axis on docetaxel (DTX) sensitivity of prostate cancer (PCa) cells. The microarray data (GSE33455) to identify differentially expressed lncRNAs and mRNAs in DTX‐resistant PCa cell lines (DU‐145‐DTX and PC‐3‐DTX) was retrieved from the Gene Expression Omnibus (GEO) database. QRT‐PCR analysis was performed to measure MALAT1 expression in DTX‐sensitive and DTX‐resistant tissues/cells. The human DTX‐resistant cell lines DU145‐PTX and PC3‐DTX were established as in vitro cell models, and the expression of MALAT1, miR‐145‐5p and AKAP12 was manipulated in DTX‐sensitive and DTX‐resistant cells. Cell viability was examined using MTT assay and colony formation methods. Cell apoptosis was assessed by TUNEL staining. Cell migration and invasion was determined by scratch test (wound healing) and Transwell assay, respectively. Dual‐luciferase assay was applied to analyse the target relationship between lncRNA MALAT1 and miR‐145‐5p, as well as between miR‐145‐5p and AKAP12. Tumour xenograft study was undertaken to confirm the correlation of MALAT1/miR‐145‐5p/AKAP12 axis and DTX sensitivity of PCa cells in vivo. In this study, we firstly notified that the MALAT1 expression levels were up‐regulated in clinical DTX‐resistant PCa samples. Overexpressed MALAT1 promoted cell proliferation, migration and invasion but decreased cell apoptosis rate of PCa cells in spite of DTX treatment. We identified miR‐145‐5p as a target of MALAT1. MiR‐145‐5p overexpression in PC3‐DTX led to inhibited cell proliferation, migration and invasion as well as reduced chemoresistance to DTX, which was attenuated by MALAT1. Moreover, we determined that AKAP12 was a target of miR‐145‐5p, which significantly induced chemoresistance of PCa cells to DTX. Besides, it was proved that MALAT1 promoted tumour cell proliferation and enhanced DTX‐chemoresistance in vivo. There was an lncRNA MALAT1/miR‐145‐5p/AKAP12 axis involved in DTX resistance of PCa cells and provided a new thought for PCa therapy.     

miR‐203 inhibits proliferation of HCC cells by targeting survivin

To validate whether down‐regulation of microRNA‐203 (miR‐203) in hepatocellular carcinoma (HCC) is involved in HCC progression by targeting survivin. MiR‐203 mimics was transfected into HepG2 cells to enhance miR‐203 expression, and miR‐203 inhibitor was transfected into HepG2 cells to inhibit miR‐203 expression. The effect of up‐regulation and down‐regulation of miR‐203 on survivin expression of HepG2 cells was evaluated using Western blot assay. The effect of miR‐203 or survivin expression on the proliferation of HepG2 cells was detected using the CKK‐8 assay. Over‐expression of miR‐203 significantly inhibited the expression of survivin in HepG2 cells (p < 0·05), and down‐expression of miR‐203 significantly promoted the expression of survivin in HepG2 cells (p < 0·05). Both over‐expression of miR‐203 and down‐regulation of survivin suppressed proliferation of HepG2 cells significantly compared with negative control. Low expression of miR‐203 contributes to the progression of HCC via targeting survivin. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

MicroRNA‐425‐5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10

Acquired chemoresistance represents a major obstacle in cancer treatment, the underlying mechanism of which is complex and not well understood. MiR‐425‐5p has been reported to be implicated tumorigenesis in a few cancer types. However, its role in regulating chemoresistance has not been investigated in colorectal cancer (CRC) cells. Microarray analysis was performed in isogenic chemosensitive and chemoresistant HCT116 cell lines to identify differentially expressed miRNAs. miRNA quantitative real‐time PCR was used to detect miR‐425‐5p expression levels between drug resistant and parental cancer cells. MiR‐425‐5p mimic and inhibitor were transfected, followed by CellTiter‐Glo^® assay to examine drug sensitivity in these two cell lines. Western Blot and luciferase assay were performed to investigate the direct target of miR‐425‐5p. Xenograft mouse models were used to examine in vivo function of miR‐425‐5p. Our data showed that expression of miR‐425‐5p was significantly up‐regulated in HCT116‐R compared with parental HCT116 cells. Inhibition of miR‐425‐5p reversed chemoresistance in HCT116‐R cells. Programmed cell death 10 (PDCD10) is the direct target of miR‐425‐5p which is required for the regulatory role of miR‐425‐5p in chemoresistance. MiR‐425‐5p inhibitor sensitized HCT116‐R xenografts to chemo drugs in vivo. Our study demonstrated that miR‐425‐5p regulates chemoresistance of CRC cells by modulating PDCD10 expression level both in vitro and in vivo. MiR‐425‐5p may represent a new therapeutic target for the intervention of CRC.     

MiR-133a Is Functionally Involved in Doxorubicin-Resistance in Breast Cancer Cells MCF-7 via Its Regulation of the Expression of Uncoupling Protein 2

The development of novel targeted therapies holds promise for conquering chemotherapy resistance, which is one of the major hurdles in current breast cancer treatment. Previous studies indicate that mitochondria uncoupling protein 2 (UCP-2) is involved in the development of chemotherapy resistance in colon cancer and lung cancer cells. In the present study we found that lower level of miR133a is accompanied by increased expression of UCP-2 in Doxorubicin-resistant breast cancer cell cline MCF-7/Dox as compared with its parental cell line MCF-7. We postulated that miR133a might play a functional role in the development of Doxorubicin-resistant in breast cancer cells. In this study we showed that: 1) exogenous expression of miR133a in MCF-7/Dox cells can sensitize their reaction to the treatment of Doxorubicin, which is coincided with reduced expression of UCP-2; 2) knockdown of UCP-2 in MCF-7/Dox cells can also sensitize their reaction to the treatment of Doxorubicin; 3) intratumoral delivering of miR133a can restore Doxorubicin treatment response in Doxorubicin-resistant xenografts in vivo, which is concomitant with the decreased expression of UCP-2. These findings provided direct evidences that the miR133a/UCP-2 axis might play an essential role in the development of Doxorubicin-resistance in breast cancer cells, suggesting that the miR133a/UCP-2 signaling cohort could be served as a novel therapeutic target for the treatment of chemotherapy resistant in breast cancer.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.     

MiR‐552 promotes the proliferation,migration and EMT of hepatocellular carcinoma cells by inhibiting AJAP1 expression

Our goal was to explore the function of miR‐552 and its potential target AJAP1 in hepatocellular carcinoma (HCC) oncogenesis and progression. In this study, bioinformatics analysis was performed to detect abnormally expressed miRNAs. The relationship between miR‐552 and AJAP1 was validated using luciferase reporter assays. RT‐qPCR and Western blot assays were applied to explore the expression level of miR‐552, AJAP1 and epithelial‐mesenchymal transition (EMT) markers. HCC cell proliferation was examined using CCK8 assays, while migration and invasion were investigated using Transwell assays. Nude mouse tumourigenesis models were established to facilitate observation of HCC progression in vivo. Finally, prognostic analysis was performed to discover how the prognosis of HCC patients correlated with miR‐552 and AJAP1 expression. MiR‐552 overexpression in HCC cells promoted HCC cell migration, invasion and EMT by targeting/suppressing AJAP1. Poorer prognosis appeared in HCC patients with higher miR‐552 expression or lower AJAP1 levels. Our findings suggested that miR‐552 promotes HCC oncogenesis and progression by inhibiting AJAP1 expression.     

MicroRNA‐214 suppresses cell proliferation and migration and cell metabolism by targeting PDK2 and PHF6 in hepatocellular carcinoma

MiR‐214 has been reported to act as a tumor suppressor or oncogene involved in various malignancies. However, the biological functions and molecular mechanisms of miR‐214 in hepatocellular carcinoma (HCC) still remain unclear. Previous studies suggest that pyruvate dehydrogenase kinase 2 (PDK2) and plant homeodomain finger protein 6 (PHF6) may be involved in some tumor cell proliferation and migration. Therefore, we studied the relationship between PDK2/PHF6 and miR‐214. The expression of miR‐214, PDK2, and PHF6 was determined by quantitative real‐time polymerase chain reaction in HCC tissues and cell lines. The Luciferase reporter assay was used to confirm the interaction between miR‐214 and PDK2/PHF6. Cell proliferation, apoptosis, and migration were evaluated by cell counting kit‐8 assay, flow cytometry, and transwell assay, respectively. The expressions levels of α‐smooth muscle actin (α‐SMA) and E‐cadherin were detected via immunofluorescence assay. Here, we found that the expression of miR‐214 decreased in HCC and was negatively correlated with PDK2 and PHF6. Moreover, PDK2 and PHF6 were the direct targets of miR‐214 in HCC cells. Functional analysis showed that knockdown of PDK2 or PHF6 as well as miR‐214 overexpression significantly suppressed cell proliferation and migration in HCC cells. Furthermore, we found that the suppression of cell proliferation and migration through PDK2 or PHF6 knockdown could be partially reversed by miR‐214 down‐regulation. Moreover, we demonstrated a decrease of mesenchymal cell marker α‐SMA and increase of the epithelial marker E‐cadherin after miR‐214 overexpression, PDK2 knockdown or PHF6 knockdown, respectively, which also suggested that cell proliferation and migration were suppressed. Additionally, lactate and pyruvic acid production experiments confirmed miR‐214 could suppress the HCC cell lactate and pyruvic acid levels by down‐regulating PDK2/PHF6. In conclusion, MiR‐214 may act as a tumor suppressor gene, presenting its suppressive role in cell proliferation and migration of HCC cells by targeting PDK2 and PHF6, and might provide a potential therapy target for patients with HCC.     

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.     

Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

Breast cancer stem cells (BCSCs) have been considered responsible for cancer progression, recurrence, metastasis and drug resistance. However, the mechanisms by which cells acquire self‐renewal and chemoresistance properties are remaining largely unclear. Herein, we evaluated the role of miR‐708 and metformin in BCSCs, and found that the expression of miR‐708 is significantly down‐regulated in BCSCs and tumour tissues, and correlates with chemotherapy response and prognosis. Moreover, miR‐708 markedly inhibits sphere formation, CD44⁺/CD24^? ratio, and tumour initiation and increases chemosensitivity of BCSCs. Mechanistically, miR‐708 directly binds to cluster of differentiation 47 (CD47), and regulates tumour‐associated macrophage‐mediated phagocytosis. On the other hand, CD47 is essential for self‐renewal, tumour initiation and chemoresistance of BCSCs, and correlates with the prognosis of breast cancer patients. In addition, the anti‐type II diabetes drug metformin are found to be involved in the miR‐708/CD47 signalling pathway. Therefore, our study demonstrated that miR‐708 plays an important tumour suppressor role in BCSCs self‐renewal and chemoresistance, and the miR‐708/CD47 regulatory axis may represent a novel therapeutic mechanism of metformin in BCSCs.     

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.