Wnt/β-Catenin activates MiR-183/96/182 expression in hepatocellular carcinoma that promotes cell invasion

Highlights

• miR-183/96/182 upregulation predicts shorter recurrence free survival in HCC patients.

• Functionally, miR-183, -96 and -182 promotes cellular invasion and migration of HCC cells.

• MiR-183, -96 and -182 concomitantly and directly targets FOXO1 3′ untranslated region.

• Over-expression of miR-183, -96 and -182 is caused by β-catenin activation in HCC.

Abstract

Nearly 50% of known miRNAs are found in clusters and transcribed as polycistronic transcripts. In this study, we showed that over-expression of miR-183/96/182 cluster is frequent in hepatocellular carcinoma (HCC), a highly aggressive malignancy that is commonly fatal. In a cohort of HCC patients (n = 81), miR-183/96/182 up-regulation correlated with metastatic features including presence of microvascular invasion, advanced tumor differentiation, and shorter recurrence-free survival. Univariate and multivariate analyses further showed miR-183/96/182 over-expression represented an independent prognostic factor (Relative Risk: 2.0471; P = 0.0289). Functional investigation using siRNA against miR-183/96/182 in two invasive HCC cells indicated significant inhibition on cell migration and invasion without affecting cell viability. Forkhead boxO1 (FOXO1) was further validated as a downstream target of these three miRNAs. In investigating the regulatory mechanism underlining miR-183/96/182 over-expression, a direct interaction of CTNNB1 on the promoter region was confirmed by ChIP-PCR and luciferase reporter validations. Knockdown of CTNNB1 also showed concordant down-regulations of miR-183, -96 and -182, and the re-expression of FOXO1. Our findings demonstrated that over-expression of miR-183/96/182 confers an oncogenic function in HCC cell dissemination, and could serve as an independent prognostic predictor for HCC patients.

Introduction

Hepatocellular carcinoma (HCC) is the most common liver malignancy that is predominant in developing countries, with approximately 700,000 deaths worldwide each year [1]. Although surgical resections and liver transplantation represent the best curative options for HCC treatment, they are only applicable to a minority of patients. This is because by the time of clinical presentation most patients are at the advanced inoperable stages with concurrent intra- and extra-hepatic metastases [2]. Since metastasis has been well regarded as importantly inferior factors on patients' survival, it is therefore of pivotal importance to understand the biologically related mechanisms of HCC metastasis.

Studies on microRNAs (miRNAs) in cancers have revealed these small non-coding RNAs can act as key posttranscriptional regulators that direct apoptosis, cell proliferation and differentiation, and cell migration [3]. In HCC, evidences supporting miRNA deregulations in correlation with tumor progression, clinicopathologic features and patient prognosis have been shown [4], [5], [6]. Taking advantage of compelling in-vivo studies on miRNAs, it was postulated that therapies targeting miRNAs could also represent a novel curative treatment for HCC [7]. Deregulation of miRNAs residing within the same cluster region is frequent in HCC [8], [9]. It is probable that several miRNAs situated on a single transcript are being controlled by the same promoter. Our earlier miRNA profiling showed miR-183, miR-96 and miR-182 belonging to the same polycistronic miRNA cluster are frequently over-expressed in HCC [8]. In this study, we further demonstrated that concordant up-regulation of miR-183/96/182 is common in HCC tumors and correlated with clinical and pathologic features that represent an independent prognostic marker for patients. In addition, our functional study also showed a role for miR-183/96/182 in promoting HCC cell dissemination through targeting Forkhead BoxO1 (FOXO1). In investigating the upstream regulatory mechanism, we found Wnt/β-catenin (CTNNB1) transcriptional activation likely underlies the increased miR-183/96/182 expressions in HCC.