Study On The Roles Of Noncoding RNAs-participated Epigenetic Regulation In The Invasion-metastasis Cascade Of Hepatocellular Carcinoma

Primary liver cancer is one of the most prevalent and lethal human cancer worldwide,particularly in Southeast Asia andAfrica. Most of primary liver cancers are hepocellularcarcinoma (HCC). Despite great advances in diagnosis and medical treatment of HCC, the5-year survival rate of HCC patients remains poor. The main causes of poor prognosis areintrahepatic and distal metastases. The molecular mechanisms contributing to HCCinitiation and development, particular HCC metastasis is not very clear at present. Previousstudies mainly focused on the genes mutation, particular those protein-coding genes andtheir functions. However, they also demonstrated that only genetic alterations could notelucidate the complexity of HCC. It is recognized that epigenetic modifications playscrucial roles in HCC. Histone acetylation modification and noncoding RNAs regulateexpressions of many important genes.In this study, we used hybridization-based microarrays, massively parallel sequencing,bioinformatic analyses and classic molecular biology technology to investigate the roles ofnoncoding RNAs in HCC. Our research has achieved the following results:The miR-200a and the level of histone H3acetylation at its promoter were reduced inhuman HCC tissues in comparison with adjacent noncancerous hepatic tissues. Usingbioinformatic analyses and previous reports, we found that the histone deacetylase4(HDAC4) could be recruited by transcription factor Sp1to the miR-200a promoter,reduced the histone H3acetylation level at the mir-200a promoter, and inhibited theexpression of miR-200a and its promoter activity. Interestingly, with in silico predictions,HDAC4was defined as a potential target of miR-200a. Using Dual-Luciferase ReporterAssay System and western blot, we confirmed that the miR-200a directly targeted the3’-untranslated region of the HDAC4mRNAand repressed expression of HDAC4.Therefore, miR-200a and HDAC4repressed each other. The HDAC4mRNAlevels weresignificantly upregulated in HCC samples in comparison with adjacent noncancerous livertissues. Histone H3acetylation levels at the mir-200a promoter and miR-200a expressionlevels were inversely correlated with HDAC4in HCC tissues. Through targeting HDAC4,miR-200a induced its own transcription and increased the histone H3acetylation level atits own promoter. Through targeting HDAC4, miR-200a also induced the upregulation oftotal acetyl-histone H3levels and increased the histone H3acetylation level at thep21WAF/Cip1promoter. Our findings suggest that the HDAC4/Sp1/miR-200a regulatory network induces the down-regulation of miR-200a, the up-regulation of HDAC4, andaberrant histone acetylation in HCC. These findings highlight a potential therapeuticapproach in targeting the HDAC4/Sp1/miR-200a regulatory network for the treatment ofHCC.The main causes of poor prognosis of HCC are intrahepatic and distal metastases.Metastases account for the vast majority of cancer-associated deaths. Although manyprotein-coding genes have been shown to regulate tumor metastases, the role of lncRNAsin metastases is unclear. The role of TGF-β-induced epithelial-mesenchymal transition(EMT) in cancer cell dissemination is well established, but the involvement of lncRNAs inTGF-β signaling is still unknown. In this study, using lncRNAs microarray, we searchedthe lncRNAs regulated by TGF-β. Recently, many RNA transcripts have been reported tofunction as competing endogenous RNAs (ceRNA) by competitively binding commonmicroRNAs. Previous reports have shown that miR-200s could inhibite EMT andup-regulate E-cadherin through direct targeting ZEB1/2. Among the regulated lncRNAs byTGF-β, we predicted76miR-200s targeting sites on46lncRNAs using the TargetScanprediction algorithm. Interestingly, one of the upregulated lncRNAs, lncRNA-Activated byTGF-β (lncRNA-ATB), which has a relatively large fold change, has three predictedmiR-200s targeting sites in relatively short span. Using RNA immunoprecipitation (RIP),affinity pull-down of endogenous miR-200s using in vitro transcribed biotin-labeledlncRNA-ATB, and Dual-Luciferase Reporter Assay System, we confirmed the specificassociation between miR-200s and lncRNA-ATB. Next, using realtime-PCR, western blot,Dual-Luciferase Reporter Assay System, and immunofluorescence staining, we found thatlncRNA-ATB upregulated ZEB1/2and induced EMT in many different cell lines. Inhuman HCC tissues lncRNA-ATB transcript level was significantly correlated withZEB1/2mRNA level, and was inversely correlated with E-cadherin mRNA level,consistent with a role of lncRNA-ATB in ZEB1/2and EMT. Using transwell invasionassay, we found that lncRNA-ATB could promote invasive behavior of HCC cells. Therole of lncRNA-ATB in ZEB1/2, EMT and cell invasion is dependent on competitivebinding of miR-200s. The lncRNA-ATB transcript level was higher in the HCC tissuesthan in the paired adjacent noncancerous hepatic tissues, and was further increased inportal vein tumor thrombus tissues. Furthermore, higher level of lncRNA-ATB wasassociated with tumor invasion in HCC patients and was inversely correlated withprognosis. Using nude mouse orthotopic tumor models, we found that lncRNA-ATB promotedintrahepatic, mesenteric, pulmonic and diaphragmatic metastases, which was notcompletely dependent on miR-200s. Next we explored the influence of lncRNA-ATB andmiR-200s on the different stages of metastasis. Using orthotopic tumor models-examiningcirculating tumor cells by flow cytometry, liver colonization models, and lung colonizationmodels, we found that lncRNA-ATB promoted intravasation of HCC cells, which wasdependent on the competitive binding of miR-200s. Also we found that lncRNA-ATBpromoted liver colonization and lung colonization of HCC cells, which was not dependenton the miR-200s. Using cDNA microarray and RIP-sequencing, we predicted thatlncRNA-ATB bound IL11mRNA, upregulated expression of IL11, induced autocrine ofIL11, and triggered STAT3signaling. We further confirmed the role of lncRNA-ATB onIL11/STAT3signaling and found that lncRNA-ATB promoted organ colonization ofdisseminated tumor cells by activating IL11/STAT3signaling.Globally, lncRNA-ATB promotes the invasion-metastasis cascade. Thus, thesefindings suggest that lncRNA-ATB, a mediator of TGF-β signaling, could predispose HCCpatients to metastases and may serve as a potential target for anti-metastatic therapies bothearly in the metastatic process and when patients present circulating tumor cells.Overall, we studied the roles and molecular mechanisms of functional noncodingRNAs in HCC initiation and development. Our researchs provide new views andlandscapes to understand the complex signaling network in HCC, and potential newstrategies for the prognosis and therapy of HCC.