Regulatory role of miRNA-1246 and Wnt/β-catenin pathway interaction in CD133+ liver cancer stem cells-driven hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), the main type of liver cancer in human, is one of the most prevalent and deadly malignancies in the world. Despite advances in therapy, prognosis remains dismal, largely attributed to our limited understanding on information related to the progressive development of the disease, particularly in their cancer-initiating and stem cell-like properties. There is increasing evidence in recent years to support the role of miRNAs in the regulation of cancer stem cell (CSC) maintenance. However, miRNAs in hepatic CSCs remain understudied. Our group has previously identified a functional subset of liver CSCs marked by the CD133 phenotype. Utilizing a comprehensive transcriptome sequencing approach, we recently compared the differential miRNA profiles of CD133+ liver CSCs and CD133- differentiated cells isolated from HCC cells Huh7 and PLC8024, and identified a significant up-regulation of miR-1246 in the CD133+ liver CSC subset. In silico prediction analysis and luciferase reporter assays found miR-1246 to target both GSK-3β and AXIN2, two key players of the Wnt/β-catenin degradation complex. This is particularly interesting as we and others have previously found the Wnt/β-catenin signaling pathway to be significantly deregulated in CD133+ liver cells as well as in HCC. miR-1246 up-regulation was further validated in additional CD133 sorted HCC cells. Expression of miR-1246 and CD133 was also found to positively correlate across a panel of liver cell lines. Functional studies were subsequently performed using lentiviral-based miR-1246 knockdown in Hep3B and Huh7. miR-1246 deregulation was closely associated with an altered ability of HCC cells to proliferate, self-renew, invade, migrate, induce capillary tube formation in endothelial cells and initiate tumor formation in vivo. Notably, reexpression of constitutively active β-catenin in HCC cells with stable miR-1246 repression rescued this altered phenotype. Knockdown of miR-1246 was associated with increased GSK-3β and AXIN2, concomitant with decreased β-catenin expression, as detected by immunofluorescence. TOP/FOP luciferase signal was also significantly down-regulated following miR-1246 suppression in both CD133-expressing Hep3B and Huh7 cells. By in vivo ubiquitylation assay, a higher level of ubiquitinated β-catenin was observed following miR-1246 knockdown in both Hep3B and Huh7 cells suggesting the decrease in β-catenin was a result of its ubiquitylation. Consistently, downstream targets of the Wnt/β-catenin pathway, including cyclin-D1, c-myc and MMP7, were also likewise decreased at the transcriptional level following miR-1246 knockdown. Taken together, we found miR-1246 to be functionally involved in driving CD133+ liver CSCs through activation of the Wnt/β-catenin and its downstream signaling cascade by directly targeting GSK-3β and AXIN2.