Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression

Abstract

Overexpression of IQGAP1 and microRNA (miRNA) dysregulation are frequent in human tumors, but little is known about the role of IQGAP1 and its relationship to miRNA in endometrial carcinogenesis. We demonstrate that IQGAP1 activates the epithelial-mesenchymal transition (EMT) program and that miR-124 directly represses IQGAP1 expression in endometrial cancer (EC) cells. The overexpression of IQGAP1 stimulates EMT features and enhances migration, invasion and proliferation of EC cells, whereas knocking down IQGAP1 expression reverses EMT and inhibits these malignant properties. Using miRNA microarray profiling, we identified 29 miRNAs (let-7b, let-7f, miR-10b, miR-15b, miR-23a, miR-24, miR-25, miR-27a, miR-29b, miR-30a-5p, miR-34a, miR-124, miR-127, miR-130b, miR-148a, miR-155, miR-191*, miR-194, miR-224, miR-362, miR-409-3p, miR-422b, miR-424, miR-453, miR-497, miR-518d, miR-518f*, miR-526a and miR-656) that are significantly down-regulated in an in vitro-selected highly invasive derivative cell line (HEC-50-HI) relative to the parental HEC-50 cells. We further identified miR-124 as a direct regulator of IQGAP1 in EC cells. Enforced expression of miR-124 suppresses EC cell invasion and proliferation. The expression of IQGAP1 mRNA was significantly elevated in EC tissues, while the expression of miR-124 was decreased. The downregulation of miR-124 correlates with a poor survival outcome for patients with EC. Treating EC cells with the demethylating agent 5-aza-2'-deoxycytidine increased miR-124 expression and down-regulated IQGAP1 levels. Our data suggest that IQGAP1 promotes EMT, migration and invasion of EC cells. MiR-124, a novel tumor suppressor miRNA that is epigenetically silenced in EC, can reverse EMT and the invasive properties, by attenuating the expression of the IQGAP1 oncogene.

Keywords: DNA methyltransferase inhibitor; IQGAP1; endometrial cancer cell invasion; epigenetics; miR-124.

Figure 1. IQGAP1 induces the epithelial-to-mesenchymal transition, invasiveness, and proliferation of endometrial cancer (EC) cells

A. Reverse transcription quantitative PCR (qPCR) analysis of IQGAP1 mRNA in the immortalized human endometrial cell line EM and the EC cells HEC-1, HEC-50, and HEC-50-HI (HI). The results are presented as the fold-change in expression compared to the EM cells. B. Western blot analysis of the IQGAP1 protein in EC cells. C, E. The expression of IQGAP1, E-cadherin, and N-cadherin proteins in HI cells transfected with control (Ctr) or IQGAP1 siRNA (C) and in HEC-1 cells expressing either the control or IQGAP1 vector (E). D, F. Phase-contrast microscopy shows the morphology of HI cells transfected with control or IQGAP1 siRNA (D) and HEC-1 cells transfected with the control or IQGAP1 vector (F). G-I. Detection of migration (G), invasion (H), and proliferation (I) in HI and HEC-1 cells after the indicated transfection. J, K. qPCR analysis of ZO-1, CK-18, and Vimentin expression in HI (J) and HEC-1 (K) cells, transfected as indicated. L. Representative images from the invasion assays.

Figure 2. MiR-124 is down-regulated in highly invasive endometrial cancer (EC) cells and directly suppresses IQGAP1 expression

A. Profiling data of down-regulated microRNAs in highly invasive HEC-50-HI (HI) cells. B. Two putative conserved miR-124-binding sites in the IQGAP1 3′ untranslated region (3′-UTR). C. Relative expression of miR-124 in immortalized human endometrial epithelial and EC cells, assessed by quantitative PCR assays. D. Expression of miR-124 in HI or HEC-1 cells transfected with a miR-124 mimic, miR-124 inhibitor, or their respective negative controls. E. Expression of the indicated mRNA and proteins in HI and HEC-1 cells after the overexpression or knockdown of miR-124. F, G. HI (F) and HEC-1 (G) cells were cotransfected with reporter plasmids containing wild-type IQGAP1 or a mutant IQGAP1 3′-UTR together with a miR-124 mimic, miR-124 inhibitor, or respective negative control. The relative luciferase activity was assayed.

Figure 3. MiR-124 maintains epithelial-like phenotypes and represses cell migration, invasion, and proliferation in endometrial cancer cells

A. Overexpression of miR-124 in HEC-50-HI (HI) cells with fibroblastic morphology converts them to an epithelial-like morphology. B. HEC-1 cells transfected with miR-124 inhibitor exhibit more mesenchymal morphology than the control cells. (A and B, phase-contrast microscopy). C-E. Migration (C), invasion (D), and proliferation (E) of HI and HEC-1 cells after the overexpression or knockdown of miR-124. F, G. Quantitative PCR analysis of the indicated genes in HI (F) and HEC-1 (G) cells after the overexpression or knockdown of miR-124, as indicated. H-J. A miR-124 mimic or its control was transfected into HI cells along with a control vector or the IQGAP1 cDNA vector lacking the 3′-UTR region. The cells were assayed for cell migration (H), invasion (I), and proliferation (J). ^** P < 0.01.

Figure 4. MiR-124 down-regulation is associated with elevated IQGAP1 expression in endometrial cancer cells

A-D. The expression levels of miR-124 (A), IQGAP1 (B), E-cadherin (C), and Vimentin (D) were assessed by a quantitative PCR analysis of 20 paired cancerous and normal tissue samples from endometrial cancer patients. E. A Kaplan-Meier survival curve of 309 TCGA (Cancer Genome Atlas database) endometrial cancer samples was created using the SurvMicro database based on the low or high risk for a poor outcome. F. Box plots demonstrating significantly lower levels of miR-124 expression in the high-risk patients.

Figure 5. MiR-124 is epigenetically silenced in endometrial cancer cells

A, B. HI and HEC-1 cells were treated with 5-aza-2′-deoxycytidine (5-AZA), Trichostatin A, or both, after which, quantitative PCR was used to measure the expression levels of miR-124 (A) and miR-34b (B). C, D. IQGAP1 protein levels (C), invasion, and proliferation (D) in endometrial cancer cells after treating with 5-AZA.