Abstract

BackgroundMetastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. Increasing evidence indicates that EMT can be regulated by microRNAs (miRNAs). The aim of this study was to investigate the role of miR-26b in modulating epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), as well as to identify its underlying mechanism of action.MethodsThe expression level of miR-26b was assessed in multiple HCC cell lines (HepG2, MHCC97H, Hep3B, MHCC97L, HCCC9810, BEL-7402, Huh7 and QGY-7703), as well as in liver tissue from patients with HCC. Follow-up studies examined the effects of a miR-26b mimic (increased expression) and a miR-26b inhibitor (decreased expression) on markers of EMT, wound healing and cell migration. The molecular target of miR-26b was also identified using a computer algorithm and confirmed experimentally.ResultsMiR-26b expression was decreased in HCC cell lines and was inversely correlated with the grade of HCC. Increased expression of miR-26b inhibited the migration and invasiveness of HCC cell lines, which was accompanied by decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal marker vimentin, at both the mRNA and protein expression levels. A binding site for miR-26b was theoretically identified in the 3′UTR of USP9X. Further studies revealed that overexpression of miR-26b repressed the endogenous level of USP9X protein expression. Overexpression of miR-26b also repressed Smad4 expression, whereas its inhibition elevated Smad4 expression.ConclusionsTaken together, our results indicate that miR-26b were inhibited in HCC. In HCC cell lines, miR-26b targeted the 3′UTR of USP9X, which in turn affects EMT through Smad4 and the TGF-β signaling pathway. Our analysis of clinical HCC samples verifies that miR-26b also targets USP9X expression to inhibit the EMT of hepatocytes. Thus, miR-26b may have some effects on the EMT of HCC cells.

Highlights

  • Metastasis is responsible for the rapid recurrence and poor survival of malignancies

  • MiR-26b downregulation correlates with progression of human hepatocellular carcinoma An increasing body of evidence indicates miR-26b is downregulated in several cancers, including hepatocellular carcinoma

  • The result of real-time PCR analysis showed that the expression level of miR-26b was markedly downregulated in all eight of the hepatocellular carcinoma (HCC) cell lines analyzed (HepG2, MHCC97L MHCC97H, BEL-7402, Huh7, HCCC-9810, Hep3B and QGY-7703), in comparison with the expression levels in normal liver tissue from three patients( P < 0.001) (Figure 1)

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Summary

Introduction

Metastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. The aim of this study was to investigate the role of miR-26b in modulating epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), as well as to identify its underlying mechanism of action. The TGF-β signaling pathway is pertinent to the current study, as it is known to play a central role in tumorigenesis and tumor progression by regulating many critical cellular processes, including cell proliferation, apoptosis and epithelial-mesenchymal transition (EMT) [7]. Subsequent studies revealed the mechanism to be the result of TGF-β-induced activation of the SNAIL transcription factor, a key mediator of EMT, and repression of epithelial markers, such as E-cadherin [12]. Loss of inactivation of Smad has been linked with multiple cancers, including pancreatic, colorectal, and gastrointestinal cancers [14,15,16]

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