Zhi-Hong Zong

MicroRNA-505 functions as a tumor suppressor in endometrial cancer by targeting TGF-α

BackgroundEndometrial carcinoma (EC) is one of the most lethal gynecologic cancers. Patients frequently have regional or distant metastasis at diagnosis. MicroRNAs are small non-coding RNAs that participate in numerous biological processes. Recent studies have demonstrated that miR-505 is associated with several types of cancer; however, the expression and function of miR-505 have not been investigated in EC.MethodsmiR-505 expression in normal endometrial tissue, endometrial carcinomas were quantified by Quantitative reverse transcription PCR. The endometrial carcinoma cell lines HEC-1B and Ishikawa were each transfected with miR-505 or scrambled mimics, after which cell phenotype and expression of relevant molecules were assayed. Dual-luciferase reporter assay and a xenograft mouse model were used to examine miR-505 and its target gene TGF-α.ResultsRT-PCR results demonstrated that miR-505 was significantly downregulated in human EC tissues compared to normal endometrial tissues. Besides, miR-505 expression was negatively associated with FIGO stage (stage I-II vs. III-IV), and lymph node metastasis (negative vs. positive). In vitro, overexpression of miR-505 significantly suppressed EC cell proliferation, increased apoptosis and reduced migratory and invasive activity. A miR-505 binding site was identified in the 3′ untranslated region of TGF-α mRNA (TGFA) using miRNA target-detecting software; a dual luciferase reporter assay confirmed that miR-505 directly targets and regulates TGFA. RT-PCR and Western-blotting results indicated that overexpressing miR-505 reduced the expression of TGF-α and the TGF-α-regulated proteins MMP2, MMP9, CDK2, while induced Bax and cleaved-PARP expression in EC cells. In vivo, overexpression of miR-505 reduced the tumorigenicity and inhibited the growth of xenograft tumors in a mouse model of EC.ConclusionsTaken together, this study demonstrates that miR-505 acts as tumor suppressor in EC by regulating TGF-α.

RhoC is a major target of microRNA-93-5P in epithelial ovarian carcinoma tumorigenesis and progression

BackgroundAn increasing amount of evidence has revealed that microRNAs regulate various biological processes, including cell differentiation, cell proliferation, apoptosis, drug resistance, and fat metabolism. Studies have shown that miR-93’s targetome in cancer has not been fully defined. Moreover, the role of miR-93 in epithelial ovarian carcinoma (EOC) remains largely unknown.MethodsMIR-93 mRNA expression in normal ovarian tissue, benign tumors, borderline tumors, primary ovarian carcinomas, and metastatic omentum was quantified. The ovarian carcinoma cell lines OVCAR3, SKOV3/DDP, and HO8910-PM were transfected with miR-93-5P, after which cell phenotype and expression of relevant molecules were assayed. Dual-luciferase reporter assay and a xenograft mouse model were used to examine miR-93 and its target gene RHOC (Ras homolog gene family member C).ResultsMIR-93 mRNA expression was significantly lower in ovarian carcinomas and borderline tumors than in normal ovarian tissues (p < 0.05), and was lower in metastatic omentum than in relative primary ovarian carcinomas (p < 0.05). MIR-93 mRNA expression was also negatively associated with differentiation (well vs. poor and moderate) and International Federation of Gynecology and Obstetrics staging (FIGO stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05), besides, miR-93 was higher expressed in mucinous adenocarcinoma than the other types (p < 0.05). MiR-93-5P overexpression reduced proliferation (p < 0.05); promoted G1 or S arrest and apoptosis (p < 0.05); suppressed migration and invasion (p < 0.05); and reduced RhoC, P70S6 kinase, Bcl-xL, matrix metalloproteinase 9 (MMP9) mRNA or protein expression; conversely, it induced P53 and cleaved PARP expression (p < 0.05). Dual-luciferase reporter assay indicated that miR-93 directly targeted RhoC by binding its 3′ untranslated region. MiR-93-5P transfection also suppressed tumor development and RhoC expression (determined by immunohistochemistry) in vivo in the xenograft mouse model (p < 0.05).ConclusionsThis is the first demonstration that miR-93-5P may inhibit EOC tumorigenesis and progression by targeting RhoC. These findings indicate that miR-93-5P is a potential suppressor of ovarian cellular proliferation. The involvement of miR-93-5P–mediated RhoC downregulation in inhibiting EOC aggressiveness may provide extended insight into the molecular mechanisms underlying cancer aggressiveness.

Upregulation of the lncRNA Meg3 induces autophagy to inhibit tumorigenesis and progression of epithelial ovarian carcinoma by regulating activity of ATG3

Maternally expressed gene 3 (Meg3), a long non-coding RNA, has been reported to be associated with the pathogenesis of multiple malignancies. However, little is known regarding the role of Meg3 in epithelial ovarian cancer (EOC). In this study, we found that the expression of Meg3 was lower in epithelial ovarian carcinoma, and has potential to be considered as a biomarker for ovarian cancer. After transfecting the ovarian cancer cell lines OVCAR3 and A2780 with Meg3, phenotypic changes and autophagy-related molecules were examined. Upregulation of Meg3 inhibited cell proliferation, plate colony formation, induced cell cycle arrest in G2 phases, and promoted apoptosis. Observation of autophagosomes was performed by transmission electron microscopy. The expression levels of LC3-II, ATG3, LAMP1 were elevated, while SQSTM1/p62 expression declined. Upregulated expression of Meg3 also suppressed tumorigenesis in vivo in a xenograft mouse model through upregulating ATG3 expression. RIP (ribonucleoprotein immunoprecipitation) and RNA pull-down assays showed that Meg3 was co-immunoprecipitated with ATG3. In addition, Meg3 protected ATG3 mRNA from degradation following treatment with actinomycin D. Overall, our results suggest that the lncRNA Meg3 acts as a tumor suppressor in EOC by regulating ATG3 activity and inducing autophagy.

microRNA 490-3P enhances the drug-resistance of human ovarian cancer cells.

BackgroundMicroRNAs (miRNAs) are non-coding, single-stranded small RNAs that regulate gene expression negatively, which is involved in fundamental cellular processes. In this study, we investigated the role of miR-490-3P in the development of drug resistance in ovarian cancer cells.MethodsThe human ovarian carcinoma cell line A2780 and A2780/Taxol were exposed to paclitaxel in the presence or absence of microRNA 490-3P transfection, after which cell viability were performed by CCK-8 assay. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting were used to assess the mRNA and protein expression levels of GST-π, MDR1 or P-gp.ResultsOur results showed higher miR-490-3P mRNA expression level in A2780/Taxol cells than in A2780 cells (p < 0.05). Following miR-490-3P transfection, both A2780 and A2780/Taxol cells showed decreased sensitivity to paclitaxel. The mRNA expression levels of MDR1, GST-π (p < 0.05) and protein expression levels of P-gp, GST-π were down-regulated after miR-490-3P transfection in comparison to mock and negative control cancer cells.ConclusionOur results demonstrate for the first time that microRNA 490-3P may be involved in the development of drug resistance in ovarian cancer.

MicroRNA-372 inhibits endometrial carcinoma development by targeting the expression of the Ras homolog gene family member C (RhoC)

Here we explore the role of microRNA-372 (miR-372) in tumorigenesis and development of endometrial adenocarcinoma (EC) and analyze the underlying mechanism. We found that miR-372 expression is much lower in EC than normal endometrial specimens. Cell function experiments demonstrated that miR-372 overexpression suppressed cell proliferation, migration, and invasion, and led to a G1 phase arrest and promoted the apoptosis of endometrial carcinoma cells in vitro. The nude mouse xenograft assay demonstrated that miR-372 overexpression suppressed tumor growth. RT-PCR and Western blot assays detected the expression of known targets of miR-372 in other malignant tumors and found Cyclin A1 and Cyclin-dependent Kinase 2 (CDK2) was downregulated by miR-372. Bioinformatic predictions and dual-luciferase reporter assays found that RhoC was a possible target of miR-372. RT-PCR and Western blot assays demonstrated that miR-372 transfection reduced the expression of RhoC, matrix metalloproteinase 2 (MMP2) and MMP9, while it increased the expression of cleaved poly (ADP ribose) polymerase (PARP) and bcl-2-associated X protein (Bax). The cell function experiments that transfected siRNA with RhoC showed the same trend as those which were transfected with miR-372. Taken together, our results demonstrated for the first time that miR-372 suppresses tumorigenesis and the development of EC; RhoC is a new and potentially important therapeutic target.

LncRNA MEG3 inhibit endometrial carcinoma tumorigenesis and progression through PI3K pathway

Long noncoding RNAs (lncRNAs) are RNA molecules more than 200 nucleotides in length that do not encode proteins. Recent studies have reported increasing numbers of functional lncRNAs. Maternally expressed gene 3 (MEG3) is a maternally imprinted gene encoding an lncRNA that plays a tumor suppressor role in various tumors. However, there has been rare report on mechanism of tumorigenesis and progression of endometrial carcinoma. In the present study, we found significantly lower MEG3 expression in endometrial carcinoma tissues than in normal endometrial tissues. MEG3 overexpression inhibited endometrial cancer cell proliferation, invasion, and metastasis; promoted apoptosis; and inhibited the activation of the phosphoinositide 3-kinase (PI3K)/m-TOR signaling pathway. RNA immunoprecipitation assay (RIP) showed that MEG3 can combine directly with PI3K. Tumor xenograft implantation in nude mice showed that MEG3 could significantly suppress tumor growth. These findings provide potential new therapeutic targets for treating endometrial cancer.

The correlation between microRNA490-3p and TGFα in endometrial carcinoma tumorigenesis and progression

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3′-untranslated region (3′ UTR). MiR-490-3p has been reported to be a suppressor in various human cancers; however, little is known about the biological functions of miR-490-3p in endometrial cancer (EC). In our study, we found that MiR-490-3p mRNA expression was significantly lower in ECs than in normal endometrial tissues. MiR-490-3p mRNA expression was also negatively associated with depth of invasion (mucosa vs. muscular and serosa) and lymph node metastasis (negative vs. positive) in EC. MiR-490-3p overexpression reduced proliferation; promoted G1 arrest and apoptosis; suppressed migration and invasion; and reduced TGFα, NF-kB, cyclin D1, survivin, matrix metalloproteinase 2 (MMP2) mRNA and protein expression, and improved Bax mRNA and protein expression. The dual-luciferase reporter assay indicated that miR-490-3p directly targeted TGFα by binding its 3′ untranslated region. MiR-490-3P transfection also suppressed tumor development and TGFα expression (as determined by immunohistochemistry and western blotting) in vivo in the xenograft mouse model. This is the first demonstration that miR-490-3P might act as a suppressor in EC tumorigenesis and progression by targeting TGFα. Our results provide a theoretical basis for the further study on the molecular target for endometrial cancer.

DLEU1 contributes to ovarian carcinoma tumourigenesis and development by interacting with miR-490-3p and altering CDK1 expression

Recently, a large number of studies have focused on the important role of long non‐coding RNAs (lncRNAs) in metabolism and development and have found that abnormal lncRNA expression is associated with the pathogenesis and development of many diseases. The lncRNA DLEU1 is involved in many solid tumours and haematological malignancies. However, its role in epithelial ovarian carcinoma (EOC) and the associated molecular mechanisms has not been reported. In this study, quantitative reverse transcription–PCR (qRT–PCR) demonstrated higher lncRNADLEU1 expression in EOC tissues than in normal tissues. Plasmid transfection of DLEU1 to up‐regulate its expression in the ovarian cancer cell lines A2780 and OVCAR3 increased cell proliferation, migration, and invasion, while inhibited apoptosis. Nude mouse xenograft assay demonstrated that DLEU1 overexpression promoted tumour growth in vivo. QRT–PCR showed decreased miR‐490‐3p expression, while Western blotting demonstrated increased its target genes CDK1, cyclinD1 and SMARCD1, as well as matrix metalloproteinase‐2 (MMP2), Bcl‐xL and P70S6K protein expression, respectively. Short interfering RNA silencing of DLEU1 produced opposite results, where qRT–PCR showed increased miR‐490‐3p expression. The dual‐luciferase reporter assay revealed a direct interaction between DLEU1 and miR‐490‐3p. MiR‐490‐3p plays a tumour suppressor role in epithelial ovarian cancer by targeting CDK1 regulation and influencing SMARCD1 and cyclin D1 (CCND1) expressions. Therefore, we suggest that through interaction with miR‐490‐3p, DLEU1 may influence the expression of CDK1, CCND1 and SMARCD1 protein, subsequently promoting the development and progression of EOC.

The role of glycogen synthase kinase-3β (GSK-3β) in endometrial carcinoma: A carcinogenesis, progression, prognosis, and target therapy marker.

Purpose Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase involved in cancer development. Herein, we demonstrated the role of GSK-3β in endometrial cancer (EC) and identified new therapeutic targets. Results GSK-3β was overexpressed in EC tissues, and was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) staging, dedifferentiation, and myometrial infiltration depth. Besides, GSK-3β overexpression predicted lower cumulative and relapse-free survival rate. si-GSK-3β transfection suppressed cell proliferation, migration, invasion, and promoted cell apoptosis through downregulating NF-kB, Cyclin D1 and MMP9 expression whereas upregulating P21 expression. Bioinformatic predictions and dual-luciferase reporter assays showed that GSK-3β was a possible target of miR-129. MiR-129 transfection reduced GSK-3β expression, and exhibited the same trend as si-GSK-3β transfection in cell function experiments. The nude mouse xenograft assay showed that miR-129 overexpression may suppress tumor growth through downregulating GSK-3β expression. Further studies showed that AZD1080, a GSK-3β inhibitor, could also inhibit EC cell proliferation, migration and invasion, while induced cell apoptosis through modulating relevant genes downstream of GSK-3β signaling. Experimental Design GSK-3β expression was determined in EC tissue and normal endometrial tissues by immunohistochemistry. After GSK-3β down-regulation by si-GSK-3β, microRNA-129 mimic transfection or GSK-3β inhibitor exposure, EC cell phenotypes and related molecules were examined. Conclusions Our results demonstrate for the first time that GSK-3β may be a novel and important therapeutic target for the treatment of endometrial carcinoma. GSK-3β inhibitor AZD1080 may be an effective drug for treating endometrial carcinoma.

Transforming growth factor-beta1 suppresses hepatocellular carcinoma proliferation via activation of Hippo signaling

In this study, we examined the expression of core proteins of the Hippo signaling pathway in hepatocellular carcinoma (HCC) cells treated with transforming growth factor-β 1(TGF-β1) and investigated the relationship between TGF-β1 and the Hippo signaling pathway, in order to better understand their roles in HCC and their potential implications for cancer therapy. We prove that the Hippo signaling pathway is involved in the TGF-β1-induced inhibition of the growth of HCC cells. Large tumor suppressor expression (LATS1) was overexpression and yes association protein 1(YAP1) translocated from the nucleus to the cytoplasm in HCC cells treated with TGF-β1. Overexpression of LATS1 and the nucleocytoplasmic translocation of YAP1 play an anti-oncogenetic role in the occurrence and development of liver cancer. Our findings provide new insight into strategies for liver cancer therapy.