Results indicated that stable overexpression of the miR-216a/217 cluster significantly promoted the migration ability of HepG2-miR-216a/217 and PLC/PRF/5-miR-216a/217 cells in vitro (Fig. 2E). These data indicated that overexpression of miR-216a/217 in
HCC with epithelial phenotypes induced EMT and enhanced migration abilities. Next, HLE cells with a mesenchymal phenotype were used as recipient cells for transfection of antagomir-miR-216a/217 (Genepharma, Shanghai, China). (Antagomirs, also known as anti-miRs or blockmirs, are a novel class of chemically engineered oligonucleotides used to silence endogenous miRNAs.) After the silencing of miR-216a/217 (Supporting Fig. 3A), striking morphological changes consistent with those of mesenchymal-to-epithelial transition (MET) were observed (Supporting Fig. 3B). Up-regulation of E-cadherin, an epithelial biomarker, and reduced expression of vimentin, a mesenchymal biomarker, 20s Proteasome activity were also observed (Supporting Fig. 3C). Furthermore, we also Vadimezan order examined the expression of miR-216a/217 on the proliferation and apoptosis of liver cancer cells. A significant increase in
cell proliferation was observed in PLC/PRF/5 at 72 hours after transfection of the p-miR-216a/217-overexpressing vector, whereas transfection of the antagomir-miR-216a/217 into HLE cells significantly decreased cell proliferation (Supporting Fig. 4A). The number of apoptotic cells (Annexin V+ cells) was not significantly affected in PLC/PRF/5 and HLE cells by modulating expression of the miR-216a/217 cluster (Supporting Fig. 4B). The recent discovery of the emergence of CSCs occurred, in part, as a result of miRNA-mediated EMT, which has provided a new avenue in understanding the regulatory mechanisms in CSCs and drug resistance. Because specific CSC markers have not been well defined for most CSCs, sphere-forming ability has emerged as a useful tool to evaluate the stemness characteristics of cells and for the
enhanced enrichment MCE of potential CSCs. Therefore, we evaluated HepG2-miR-216a/217 and PLC/PRF/5-miR-216a/217 cells for their ability to form tumor spheres. It was observed that HepG2-miR-216a/217 and PLC/PRF/5-miR-216a/217 generated 2∼3-fold more spheres than corresponding control cells (Fig. 3A,B). Flow cytometric analysis further demonstrated that sphere-forming cells derived from HepG2-miR-216a/217 and PLC/PRF/5-miR-216a/217 cells gave an enriched epithelial cell adhesion molecule (EpCAM)+ cell subpopulation, consistent with reported characteristics of liver CSCs[15] (Fig. 3C,D). The parental HepG2 had a small percentage of EpCAM+ cell subpopulation (12.6%), which was increased to 23.9% after transfection with miR-216a/217 (Fig. 3C). This suggests that the miR-216a/217 cluster may play an important role in regulating the stem-like traits of HCC cells by inducing EMT.