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MiR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma.

ABSTRACT: Epithelial-to-mesenchymal transition (EMT) is associated with poor prognosis and metastasis in hepatocellular carcinoma. We have previously demonstrated an in vivo model of liver cancer in which mesenchymal cells post-EMT demonstrate a high rate of invasive growth and metastasis. Here, we investigate the role of microRNA 200 (miR-200) family members and epigenetic modifications on the maintenance of mesenchymal/metastatic phenotype after EMT. Mesenchymal cells post-EMT demonstrates high levels of E-box repressors Zeb1 and Zeb2 and downregulation of four miR-200 family members (miR-200a, miR-200b, miR-200c and miR-429). In addition, DNA sequencing after bisulfite modification demonstrates that several CpG sites within the E-cadherin promoter are methylated in mesenchymal cells. In mesenchymal cells, forced expression of miR-200b results in a significant increase in E-cadherin and a reduction in cell migration/invasion. Despite these mesenchymal-to-epithelial transition (MET) changes in vitro, there is no significant change in metastatic potential after miR-200b upregulation in vivo. After the mesenchymal cells were treated with combination of DNA methyltransferase (DNMT) inhibitor and upregulation of miR-200b, invasive phenotype was significantly reduced and metastatic potential was eliminated. Direct targeting of E-cadherin with short hairpin RNA does not restore metastatic potential after DNMT inhibition and miR-200b re-expression. In addition, restoration of E-cadherin alone was unable to block metastatic potential in primary mesenchymal cells. In conclusion, targeting mesenchymal liver cancer cells with miR-200b and DNMT inhibitor reduces metastatic potential irrespective of E-cadherin expression. Thus, the broader differentiation and MET effects of DNMT inhibition and miR-200b must be considered in terms of rescuing metastatic potential.

SUBMITTER: Ding W

PROVIDER: S-EPMC3412647 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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miR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma.

Ding W W Dang H H You H H Steinway S S Takahashi Y Y Wang H-G HG Liao J J Stiles B B Albert R R Rountree C B CB

Oncogenesis 20120611

Epithelial-to-mesenchymal transition (EMT) is associated with poor prognosis and metastasis in hepatocellular carcinoma. We have previously demonstrated an in vivo model of liver cancer in which mesenchymal cells post-EMT demonstrate a high rate of invasive growth and metastasis. Here, we investigate the role of microRNA 200 (miR-200) family members and epigenetic modifications on the maintenance of mesenchymal/metastatic phenotype after EMT. Mesenchymal cells post-EMT demonstrates high levels o ...[more]

PMID: 23552699

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Project description:BackgroundY-box binding protein 1 (YB1 or YBX1) plays a critical role in tumorigenesis and cancer progression. However, whether YB1 affects malignant transformation by modulating non-coding RNAs remains largely unknown. This study aimed to investigate the relationship between YB1 and microRNAs and reveal the underlying mechanism by which YB1 impacts on tumor malignancy via miRNAs-mediated regulatory network.MethodsThe biological functions of YB1 in hepatocellular carcinoma (HCC) cells were investigated by cell proliferation, wound healing, and transwell invasion assays. The miRNAs dysregulated by YB1 were screened by microarray analysis in HCC cell lines. The regulation of YB1 on miR-205 and miR-200b was determined by quantitative real-time PCR, dual-luciferase reporter assay, RNA immunoprecipitation, and pull-down assay. The relationships of YB1, DGCR8, Dicer, TUT4, and TUT1 were identified by pull-down and coimmunoprecipitation experiments. The cellular co-localization of YB1, DGCR8, and Dicer were detected by immunofluorescent staining. The in vivo effect of YB1 on tumor metastasis was determined by injecting MHCC97H cells transduced with YB1 shRNA or shControl via the tail vein in nude BALB/c mice. The expression levels of epithelial to mesenchymal transition markers were detected by immunoblotting and immunohistochemistry assays.ResultsYB1 promoted HCC cell migration and tumor metastasis by regulating miR-205/200b-ZEB1 axis partially in a Snail-independent manner. YB1 suppressed miR-205 and miR-200b maturation by interacting with the microprocessors DGCR8 and Dicer as well as TUT4 and TUT1 via the conserved cold shock domain. Subsequently, the downregulation of miR-205 and miR-200b enhanced ZEB1 expression, thus leading to increased cell migration and invasion. Furthermore, statistical analyses on gene expression data from HCC and normal liver tissues showed that YB1 expression was positively associated with ZEB1 expression and remarkably correlated with clinical prognosis.ConclusionThis study reveals a previously undescribed mechanism by which YB1 promotes cancer progression by regulating the miR-205/200b-ZEB1 axis in HCC cells. Furthermore, these results highlight that YB1 may play biological functions via miRNAs-mediated gene regulation, and it can serve as a potential therapeutic target in human cancers.

Project description:Inflammatory bowel disease (IBD), which consists of Crohn's disease (CD) and ulcerative colitis (UC), is a chronic, inflammatory disorder of the gastro-intestinal tract with unknown etiology. Current evidence suggests that intestinal epithelial cells (IECs) is prominently linked to the pathogenesis of IBD. Therefore, maintaining the intact of epithelium has potential roles in improving pathophysiology and clinical outcomes of IBD. MicroRNAs (miRNAs) act as post-transcriptional gene regulators and regulate many biological processes, including embryonal development, cell differentiation, apoptosis and proliferation. In this study, we found that miR-200b decreased significantly in inflamed mucosa of IBD, especially for UC, when compared with their adjacent normal tissue. Simultaneously, we also found that the genes of E-cadherin and cyclin D1 were reduced significantly and correlated positively to the miR-200b. In addition, the upregulation of transforming growth factor-beta 1 (TGF-?1) was inversely correlated to the miR-200b in IBD. To investigate the possible roles of miR-200b in IECs maintaining, we used TGF-?1 to induce epithelial-mesenchymal transition (EMT) in IEC-6 initially. After sustained over-expressing miR-200b in IEC-6, the EMT was inhibited significantly that was characterized by downregulation of vimentin and upregulation of E-cadherin. Furthermore, we found that miR-200b enhanced E-cadherin expression through targeting of ZEB1, which encode transcriptional repressors of E-cadherin. SMAD2 was found to act as a target of miR-200b with direct evidence that miR-200b binding to the 3' UTR of SAMD2 and the ability of miR-200b to repress SMAD2 protein expression. With SMAD2 depletion, the expression of vimentin decreased correspondingly, which suggested miR-200b might reduce vimentin through regulating the SMAD2. With endogenous over-expression of miR-200b, the proliferation of IEC-6 cells increased significantly by increasing S-phase entry and promoting expression of the protein cyclin D1. Summarily, our study suggested a potential role for mir-200b in maintaining intact of intestinal epithelium through inhibiting EMT and promoting proliferation of IECs.

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MiR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma.

Publications

miR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma.

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