microRNA‐145‐3p inhibits non‐small cell lung cancer cell migration and invasion by targeting PDK1 via the mTOR signaling pathway

The mammalian target of rapamycin (mTOR) pathway is dysregulated in more than 50% of all human malignancies and is a major target in cancer treatment. In this study, we explored the underlying mechanism involving microRNA‐145‐3p (miR‐145‐3p) in the development and progression of non‐small cell lung cancer (NSCLC) by targeting PDK1 via the mTOR signaling pathway. NSCLC tissues and adjacent normal tissues were obtained from 83 NSCLC patients. miR‐145‐3p, PDK1, and mTOR levels were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry. Human NSCLC cell lines A549 and H1299 were transfected with miR‐145‐3p and siPDK1 to confirm the effect of miR‐145‐3p and PDK1 on NSCLC cells in vitro. Cell growth was evaluated by a CCK8 assay. Cell motility and chemotaxis analysis were determined by the scratch test and chemotaxis assay, respectively. The protein levels of PDK1 and mTOR were measured using the western blotting. Results showed lower level of miR‐145‐3p and higher levels of PDK1 and mTOR in NSCLC tissues compared to the adjacent normal tissues. In vitro results showed that cell growth, cell motility, and chemotaxis were all inhibited in cells transfected with miR‐145‐3p and those transfected with siPDK. Additionally, dual luciferase reporter gene assay helped confirmed that PDK1 is a target of miR‐145. Finally, levels of PDK1, mTOR, and phosphorylated‐mTOR were lower in cells transfected with miR‐145‐3p as well as those with siPDK1. These findings indicate that miR‐145‐3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppressing the mTOR pathway.     

circEPSTI1 regulates ovarian cancer progression via decoying miR‐942

Increasing studies show that circular RNAs (circRNAs) play vital roles in tumour progression. But, how circRNAs function in ovarian cancer is mostly unclear. Here, we detected the expression of circEPSTI1 in ovarian cancer and explored the function of circEPSTI1 in ovarian cancer via a series of experiments. Then, we performed luciferase assay and RNA immunoprecipitation (RIP) assay to explore the competing endogenous RNA (ceRNA) function of circEPSTI1 in ovarian cancer. qRT‐PCR verified that circEPSTI1 was overexpressed in ovarian cancer. Inhibition of circEPSTI1 suppressed ovarian cancer cell proliferation, invasion but promoted cell apoptosis. Luciferase assays and RIP assay showed that circEPSTI1 and EPSTI1 (epithelial stromal interaction 1) could directly bind to miR‐942. And circEPSTI1 could regulate EPSTI1 expression via sponging miR‐942. In summary, circEPSTI1 regulated EPSTI1 expression and ovarian cancer progression by sponging miR‐942. circEPSTI1 could be used as a biomarker and therapeutic target in ovarian cancer.     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.     

Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

AFAP1‐AS1 is a long non‐coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1‐AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1‐AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp‐2 cells. Indeed, we found not only that AFAP1‐AS1 was up‐regulated in laryngeal carcinoma specimens and cells, but also that stemness‐associated genes were overexpressed. Silencing of AFAP1‐AS1 promoted HEp‐2 cell chemoresistance under cisplatin treatment. Expression of AFAP1‐AS1 was increased in drug‐resistant Hep‐2 cells. We then probed the mechanism of AFAP1‐AS1 activity and determined that miR‐320a was a potential molecular target of AFAP1‐AS1. Luciferase reporter and qRT‐PCR assays of AFAP1‐AS1 and miR‐320a levels in human specimens and cell cultures indicated that AFAP1‐AS1 negatively regulates miR‐320a. To discover the molecular mechanism of miR‐320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3′‐untranslated regions (3′‐UTR) of RBPJ into a luciferase reporter, we determined that miR‐320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1‐AS1 increases RBPJ expression by negatively regulating miR‐320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1‐AS1 silencing. Taken together, these results suggest that AFAP1‐AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR‐320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.     

microRNA‐124 inhibits stem‐like properties and enhances radiosensitivity in nasopharyngeal carcinoma cells via direct repression of expression of JAMA

Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA‐124 (miR‐124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR‐124 in NPC CSCs. qRT‐PCR was performed to measure miR‐124 expression in NPC tissues and cell lines and the effects of miR‐124 on stem‐like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR‐124 with the 3′UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR‐124 in an animal model and clinical samples. Down‐regulation of miR‐124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR‐124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up‐regulation of miR‐124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR‐124 can inhibit stem‐like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.     

CircRhoC promotes tumorigenicity and progression in ovarian cancer by functioning as a miR‐302e sponge to positively regulate VEGFA

Ovarian cancer is a leading cause of deaths due to gynaecological malignancy. While endogenous non‐coding circular RNAs (circRNAs) in cancer have attracted attention, their roles in ovarian cancer are not known. We used qRT‐PCR to quantify expression of circRhoC in ovarian cancer tissues and normal tissues. The effects of overexpressing or destruction of circRhoC on the phenotype of ovarian cancer cells were assessed both in vitro and in vivo. Dual‐luciferase reporter assay assesses the microRNA sponge function of circRhoC. Western blotting was used to confirm the effects of circRhoC and microRNA on target gene expression. Our results showed that circRhoC was significantly up‐regulated in ovarian cancer tissues compared to normal ovarian tissues. Overexpression of circRhoC in CAOV3 ovarian cancer cell increased cell viability, migration and invasion ability; destroying circRhoC in A2780 had the opposite effects and inhibited ovarian tumour cell A2780 dissemination in the peritoneum in vivo. We confirmed circRhoC functions as a sponge for miR‐302e to positively regulate VEGFA; FISH experiments showed that circRhoC could co‐focal with miR‐302e; besides, overexpression of miR‐302e reversed the ability of circRhoC to positively regulate VEGFA, and what's more, RIP assay showed that circRhoC could directly bind with VEGFA; besides, VEGFA expression level in ovarian cancer tissues was positively associated with circRhoC expression. In conclusion, the oncogenic effect of RhoC in ovarian cancer is at least in part due to circRhoC, which functions not only as a miR‐302e sponge to positively regulate VEGFA protein expression, but may also directly bind and modulate VEGFA expression.     

miR‐150‐5p suppresses the stem cell‐like characteristics of glioma cells by targeting the Wnt/β‐catenin signaling pathway

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR‐150‐5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR‐150‐5p and catenin beta 1 (CTNNB1, which encodes β‐catenin) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. The expression levels of downstream genes of the Wnt/β‐catenin pathway and stem cell markers were detected by qRT‐PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR‐150‐5p and β‐catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR‐150‐5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR‐150‐5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β‐catenin pathway and stem cell markers. Furthermore, miR‐150‐5p expression was decreased and β‐catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR‐150‐5p contributed to CD133? cells with stem cell‐like phenotype, whereas overexpression of miR‐150‐5p suppressed CD133+ glioma stem cell‐like characteristics. In conclusion, miR‐150‐5p inhibited the progression of glioma by controlling stem cell‐like characteristics via regulating the Wnt/β‐catenin pathway, providing a novel target for glioma treatment.     

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‐10b expression in breast cancer stem cells supports self‐renewal through negative PTEN regulation and sustained AKT activation

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT–PCR analysis. One of the most altered miRNAs was miR‐10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR‐10b in MCF‐7 cells (miR‐10b‐OE cells) promoted higher self‐renewal and expression of stemness and epithelial–mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR‐10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self‐renewal. Bioinformatics analyses identified several potential miR‐10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self‐renewal. The targeting of PTEN by miR‐10b was confirmed using a luciferase reporter, qRT–PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR‐10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self‐renewal ability of CSCs and breast cancer cell lines overexpressing miR‐10b. In conclusion, miR‐10b regulates the self‐renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.     

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.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.     

MiR‐30c protects diabetic nephropathy by suppressing epithelial‐to‐mesenchymal transition in db/db mice

Epithelial‐to‐mesenchymal transition (EMT) plays a significant role in tubulointerstitial fibrosis, which is a hallmark of diabetic nephropathy. Thus, identifying the mechanisms of EMT activation could be meaningful. In this study, loss of miR‐30c accompanied with increased EMT was observed in renal tubules of db/db mice and cultured HK2 cells exposed to high glucose. To further explore the roles of miR‐30c in EMT and tubulointerstitial fibrosis, recombinant adeno‐associated viral vector was applied to manipulate the expression of miR‐30c. In vivo study showed that overexpression of miR‐30c suppressed EMT, attenuated renal tubulointerstitial fibrosis and reduced proteinuria, serum creatinine, and BUN levels. In addition, Snail1 was identified as a direct target of miR‐30c by Ago2 co‐immunoprecipitation, luciferase reporter, and Western blot assays. Downregulating Snail1 by siRNA reduced high glucose‐induced EMT in HK2 cells, and miR‐30c mimicked the effects. Moreover, miR‐30c inhibited Snail1‐TGF‐β1 axis in tubular epithelial cells undergoing EMT and thereby impeded the release of TGF‐β1; oppositely, knockdown of miR‐30c enhanced the secretion of TGF‐β1 from epitheliums and significantly promoted proliferation of fibroblasts and fibrogenesis of myofibroblasts, aggravated tubulointerstitial fibrosis, and dysfunction of diabetic nephropathy. These results suggest a protective role of miR‐30c against diabetic nephropathy by suppressing EMT via inhibiting Snail1‐TGF‐β1 pathway.     

miR‐542‐3p prevents ovariectomy‐induced osteoporosis in rats via targeting SFRP1

Secreted frizzled‐related protein‐1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR‐542‐3p and sFRP1 expression by RNA sequencing and qRT‐PCR. In addition, there was a significant negative correlation between miR‐542‐3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR‐542‐3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR‐542‐3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR‐542‐3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast‐induced mesenchymal stem cells (MSC), whereas the expression of miR‐542‐3p was significantly decreased. And miR‐542‐3p transfected MSCs showed a significant increase in osteoblast‐specific marker expression, indicating that miR‐542‐3p is necessary for MSC differentiation. Inhibition of miR‐542‐3p reduced bone formation, confirmed miR‐542‐3p play a role in bone formation in vivo. In general, these data suggest that miR‐542‐3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.     

Hsa‐miR‐132 inhibits proliferation of hepatic carcinoma cells by targeting YAP

MicroRNAs and Yes‐associated protein (YAP) play an important role in the occurrence and development of hepatic carcinomas. However, the interaction between microRNAs and YAP was seldom elucidated. In this study, we showed that miR‐132 could target YAP gene by using dual‐luciferase reporter system. Further quantitative polymerase chain reaction analysis and western blotting showed that miR‐132 could significantly reduce the expression of YAP at mRNA and protein levels. Results of annexin V‐fluorescein isothiocyanate, 5‐ethynyl‐2′‐deoxyuridine staining and transwell assays showed that miR‐132 significantly promoted the cell apoptosis and effectively inhibited the proliferation and invasion of hepatoma cells. These results indicated that miR‐132 could inhibit the growth of hepatoma cells by targeting YAP gene and reducing its expression level. Taken together, results from this study would help us to understand the mechanisms for occurrence and development of hepatic carcinoma and provide new targets for diagnosis and treatment of liver cancer. Copyright © 2015 John Wiley & Sons, Ltd.     

MicroRNA‐1258, regulated by c‐Myb,inhibits growth and epithelial‐to‐mesenchymal transition phenotype via targeting SP1 in oral squamous cell carcinoma

The biological function and underlying mechanism of miR‐1258 has seldom been investigated in cancer progression, including in oral squamous cell carcinoma (OSCC). In the current study, we revealed that the expression level of miR‐1258 was significantly down‐regulated in OSCC tissues and cell lines. Restoration of miR‐1258 decreased OSCC cell growth and invasion. The luciferase and Western blot assays revealed that SP1 protein was a downstream target of miR‐1258. Overexpression of SP1 dismissed miR‐1258’s effect on cell growth and invasion. We also revealed that c‐Myb inhibited miR‐1258 by directly binding at its promoter. In addition, miR‐1258 inhibited PI3K/AKT and ERK signalling pathway activity. Taken together, these findings demonstrated that miR‐1258 may function as a tumour‐suppressive micorRNA in OSCC and suggested that miR‐1258 may be a potential therapeutic target for OSCC patients.     

Upregulation of miR‐874‐3p and miR‐874‐5p inhibits epithelial ovarian cancer malignancy via SIK2

Based on miR‐874 expression levels in the GSE47841 microarray, we hypothesized that the mature products of miR‐874, miR‐874‐3p, or miR‐874‐5p, would inhibit epithelial ovarian cancer (EOC) cell proliferation, metastasis, and chemoresistance. We first examined miR‐874‐3p and miR‐874‐5p expression levels in primary EOC tumor tissue samples and found that they were significantly decreased. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) cell proliferation and transwell assays revealed that miR‐874‐3p and miR‐874‐5p significantly inhibit EOC cell proliferation, migration, and invasion. Then, using MTT and soft agar assays of paclitaxel‐treated Caov3 and SKOV3 cells transfected with miR‐874‐3p and miR‐874‐5p, we found that miR‐874‐3p and miR‐874‐5p enhance EOC cell chemosensitivity. We then confirmed that serine/threonine‐protein kinase 2 (SIK2) was a target gene of miR‐874‐3p and miR‐874‐5p. Overall, the results of this study indicate that SIK2 expression can serve as a prognostic biomarker for EOC and that miR‐874‐3p and miR‐874‐5p have the potential to enhance clinical treatment of EOC.