MicroRNA-194 inhibits epithelial to mesenchymal transition of endometrial cancer cells by targeting oncogene BMI-1

Abstract

Background: Epithelial-mesenchymal transition (EMT) is the key process driving cancer metastasis. Oncogene/self renewal factor BMI-1 has been shown to induce EMT in cancer cells. Recent studies have implied that noncoding microRNAs (miRNAs) act as crucial modulators for EMT. The aims of this study was to determine the roles of BMI-1 in inducing EMT of endometrial cancer (EC) cells and the possible role of miRNA in controlling BMI-1 expression.

Methods and results: We evaluated the expression of BMI-1 gene in a panel of EC cell lines, and detected a strong association with invasive capability. Stable silencing of BMI-1 in invasive mesenchymal-type EC cells up-regulated the epithelial marker E-cadherin, down-regulated mesenchymal marker Vimentin, and significantly reduced cell invasion in vitro. Furthermore, we discovered that the expression of BMI-1 was suppressed by miR-194 via direct binding to the BMI-1 3'-untranslated region 3'-UTR). Ectopic expression of miR-194 in EC cells induced a mesenchymal to epithelial transition (MET) by restoring E-cadherin, reducing Vimentin expression, and inhibiting cell invasion in vitro. Moreover, BMI-1 knockdown inhibited in vitro EC cell proliferation and clone growth, correlated with either increased p16 expression or decreased expression of stem cell and chemoresistance markers (SOX-2, KLF4 and MRP-1).

Conclusion: These findings demonstrate the novel mechanism for BMI-1 in contributing to EC cell invasion and that repression of BMI-1 by miR-194 could have a therapeutic potential to suppress EC metastasis.

Figure 1

Association between BMI-1 expression and invasive potential and EMT features of EC cell lines. A. BMI-1 protein levels in the panel of EC cell lines were determined by Western blot analysis. B. Invasive ability of EC cells was evaluated by Matrigel invasion assays. Data represent the mean ± SE of three independent experiments. C. Highly invasive HEC-50B-HI cells exhibiting fibroblastic morphology and the parental HEC-50B cells showing epithelial-like appearance were fixed with neutral-buffered formalin, stained in Giemsa, and images were taken (magnification × 100). D. Western blot analysis of epithelial marker E-cadherin and mesenchymal marker Vimentin in HEC-50B and HEC-50B-HI cells.

Figure 2

Silencing of BMI-1 expression reverts EMT phenotype and reduces EC cell invasion. A. Comparison of BMI-1 levels in un-transfected HEC-50B-HI, HEC-50B-HI-BMI-1 shRNA and control shRNA cells. B. Morphological changes of HEC-50B-HI cells after knockdown of BMI-1 expression 27 (magnification × 100). C. Effects of BMI-1 suppression on cell invasion ability using Matrigel invasion assay. Data represent the mean ± SE of three independent experiments. *P < 0.01. D. Western blots of epithelial marker E-cadherin and mesenchymal marker Vimentin in un-transfected HEC-50B-HI, HEC-50B-HI-BMI-1 shRNA and control shRNA cells.

Figure 3

MiR-194 directly targets BMI-1, and reverses invasive, EMT phenotype in EC cells. A. Sequence of miR-194 binding site in the BMI-1 3'UTR predicted with TargetScan. B. MiR-194 expression levels in HEC-50B and HEC-50B-HI cells were determined by Real-time RT-PCR. Data represent the mean ± SE of three independent experiments. *P < 0.01. C. Western blotting analysis of BMI-1, E-cadherin and Vimentin in un-transfected EC cells and EC cells transfected with miR-194 or control miRNA. D. HEC-50B-HI and HHUA cells were transfected with WT or MT BMI-1 3'UTR luciferase vectors, along with miR-194, miR-128 or control miRNA. Luciferase activity was measured at 24 h after transfection. Data represent the mean ± SE of three independent experiments. *P < 0.01. E. Invasive potential of un-transfected and EC cells transfected with miR-194 or control miRNA was evaluated by Matrigel invasion assays. Data represent the mean ± SE of three independent experiments. *P < 0.01.

Figure 4

Knockdown of BMI-1 repressed in vitro cell proliferation and clone growth. A. Effects of BMI-1 repression on cell proliferation assessed by MTT assay in un-transfected HEC-50B-HI cells, HEC-50B-HI-BMI-1 shRNA and control shRNA cells. *P < 0.05. B. Colony formation was assessed following stable repression of BMI-1. *P < 0.05. C. Western immunoblot analysis of p16 protein expression in un-transfected HEC-50B-HI cells, HEC-50B-HI-BMI-1 shRNA cells and control shRNA cells. D. mRNA expression levels of SOX-2, KLF4 and MRP-1 in HEC-50B-HI-BMI-1 shRNA and control shRNA cells were determined by Real-time RT-PCR. Data represent the mean ± SE of three independent experiments.