MiR-101 suppresses the epithelial-to-mesenchymal transition by targeting ZEB1 and ZEB2 in ovarian carcinoma

Abstract

Ovarian carcinoma is the most lethal gynecologic malignancy; the majority of patients succumb to the disease within 5 years of diagnosis. The poor survival rate is attributed to diagnosis at advanced stage, when the tumor has metastasized. The epithelial-to-mesenchymal transition (EMT) is a necessary step toward metastatic tumor progression. Through integrated computational analysis, we recently identified a master microRNA (miRNA) network that includes miR-101 and regulates EMT in ovarian carcinoma. In the present study, we characterized the functions of miR-101. Using reporter gene assays, we demonstrated that miR-101 suppressed the expression of the E-cadherin repressors ZEB1 and ZEB2 by directly targeting the 3'-untranslated region (3'UTR) of both ZEB1 and ZEB2. Introduction of miR-101 significantly inhibited EMT and cell migration and invasion. Introducing cDNAs of ZEB1 and ZEB2 without 3'UTR abrogated miR-101-induced EMT alteration, respectively. Our findings showed that miR-101 represents a redundant mechanism for the miR-200 family that regulates EMT through two major E-cadherin transcriptional repressors.

Figure 1

miR-101 directly targets ZEB1 and ZEB2. (A) The two predicted miR-101-binding sites in the ZEB1 3′-UTR region are shown in the upper panel and the one predicted miR-101-binding site in the ZEB2 3′-UTR region is shown in the lower panel. (B) The pmirGLO-ZEB1 and pmirGLO-ZEB2 reporter genes had putative miR-101-binding sites cloned into the pmirGLO-control vector. The pmirGLO-ZEB1-mt vector had the two miR-101-binding sites deleted; the deletion was confirmed by sequencing. The pmirGLO-ZEB2-mt vector had the one miR-101-binding site deleted, and again the deletion was confirmed by sequencing. HeLa cells were transfected with pmirGLO-ZEB1 or pmirGLO-ZEB1-mt or with pmirGLO-ZEB2 or pmirGLO-ZEB2-mt, together with an miR-101 mimic or mimic negative control. The mean relative luciferase activities are shown, with standard errors, from 3 independent experiments.

Figure 2

miR-101 suppresses endogenous ZEB1 and ZEB2 expression. (A) ZEB1 and ZEB2 in SKOV3 cells were quantified by TaqMan real-time PCR. All data are means of triplicate PCR assays. (B) Levels of ZEB1 and ZEB2 proteins in SKOV3 cells transfected with miR-101 mimic (miR-101) or control (miR-Ctrl) were measured by western blotting using whole cell lysates from each sample.

Figure 3

miR-101 suppresses EMT and cell migration and invasion. (A) Levels of E-cadherin, fibronectin, N-cadherin and vimentin proteins in SKOV3 cells after transfection with miR-101 mimic (miR-101) or control (miR-Ctrl) were measured by western blotting using whole cell lysates from each sample. Actin was used to control protein loading. (B) Wound-healing assay was performed in SKOV3 cells. The extent of closure of the wound, representing cell migration, was monitored under phase-contrast microscopy and images were captured at 0 and 16 h. (C and D) Cell invasion assays in SKOV3 cells transfected with miR-101 or miR-Ctrl after 48 h. The cells were allowed to invade toward the lower chamber for 22 h. Each result in (D) represents an average of triplicates. (E) E-cadherin and F-actin immunofluorescence staining of SKOV3 cells transfected with miR-101 or miR101-Ctrl for 72 h. EMT, epithelial-to-mesenchymal transition.

Figure 4

ZEB1 and ZEB2 are EMT enhancers. (A and B) Levels of E-cadherin protein in SKOV3 cells after transfection with si-ZEB1, si-ZEB2, or siRNA-ctrl were measured by western blotting using whole cell lysates from each sample. Actin was used to control protein loading. (C) Wound-healing assay in SKOV3 cells after transfection with si-ZEB1, si-ZEB2, or si-ZEB1+2; the extent of closure of the wound, representing cell migration, was monitored under phase-contrast microscopy and images were captured at 0 and 16 h. EMT, epithelial-to-mesenchymal transition.

Figure 5

miR-101 suppresses EMT and increases E-cadherin expression independent of the miR-200 family. (A) Levels of E-cadherin (E-cad) and ZEB1 proteins in empty vector (EV) or ZEB1-overexpressing (C5 and C10, which are different clones) SKOV3 cells after transfection with miR-101 mimic (miR-101) or control (miR-Ctrl) were measured by western blotting using whole cell lysates from each sample. (B) Levels of E-cadherin and ZEB2 proteins in EV or ZEB2-overexpressing (C3 and C7, which are different clones) SKOV3 cells after transfection with miR-101 of miR-Ctrl were measured by western blotting using whole cell lysates from each sample. Actin was used to control protein loading. (C) Levels of miR-200a, miR-200c, and miR-141 in SKOV3 cells after miR-101 or miR-Ctrl transfection were analyzed by TaqMan miRNA quantitative PCR at 24, 48, 72 and 96 h. EMT, epithelial-to-mesenchymal transition.