Mir-101Suppresses Epithelial-to-mesenchymal Transition By Targeting ZEB1and ZEB2in Serous Ovarian Carcinoma

Ovarian carcinoma is the most lethal gynecologic malignancy; the majority of patients die within5years 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-101and regulates EMT in Ovarian carcinoma. In this study, we characterized the functions of miR-101. Using reporter gene assays, we demonstrated that miR-101suppressed the expression of E-cadherin repressors ZEB1and ZEB2by directly targeting the3’-untranslated region of both ZEB1and ZEB2. Introduction of miR-101significantly inhibited EMT and cell migration and invasion. Introducing cDNA of ZEB1and ZEB2without3’UTR abrogated miR-101-induced EMT alteration respectively. Our findings revealed that miR-101represents a redundant mechanism for the miR-200family that regulates EMT through two major E-cadherin transcriptional repressors. This study is ivided to5parts:1. We took an integrated approach and analyzed mRNA, miRNA, DNA copy number, and DNA methylation data from459serous OvCa cases in TCGA to uncover the underlying molecular network of mesenchymal subtype (iM) of ovarian cancer. We screened7serous OvCa cell lines (OVCA420, OVCA429, OVCA432, OVCA433, OVCAR3, SKOV3and HeyA8) for miR-101levels to see if miR-101expression is positively related with E-cadherin.2. We transfected SKOV3cells with either miR-101or miR-Ctrl to determine whether forced expression of miR-101can promote the epithelial phenotype and inhibit migration and invasion in ovarian cancer cell lines.3. To confirm that miR-101specifically regulates ZEB1and ZEB2through the predicted binding sites, we cloned the3’-UTR of ZEB1or the3’-UTR of ZEB2into the pmirGLO-ctrl vector to generate pmirGLO-ZEB1and pmirGLO-ZEB2constructs. We also generated the constructs pmirGLO-ZEB1-mt and pmirGLO-ZEB2-mt, from which the miR-101-binding site sequences on the3’-UTR of ZEB1or ZEB2were deleted.4. We knocked down ZEB1or ZEB2by two different siRNAs to determine whether ZEB1or ZEB2low expression could inhibit EMT in ovarian cancer cells. We transfected SKOV3cells with pcDNA3.1(+)-ZEB1or pcDNA3.1(+)-ZEB2that did not contain a3’-UTR to obtain stable clones. We transfected these clones with miR-101to determine whether miR-101’s inhibition of EMT was mediated by ZEB1or ZEB2.The conclusion drawing from the present study is as follows:1. Integrated genomic analyses from The Cancer Genome Atlas (TCGA) data divides serous ovarian cancer into2categories, integrated mesenchymal subtype (iM) and integrated epithelial subtype (iE). iM was associated with poor overall survival. Eight key miRNAs (miR-25, miR-506, miR-29c, miR-182, miR-128, miR-101, miR-141, and miR-200a), which were downregulated in iM than iE, were predicted to regulate89%of the miRNA-associated genes. Lower expression of miR-101in iM of serous OvCa is related to copy number status for miR-101. Epithelial ovarian cancer cell lines had significantly higher miR-101expression compared with mesenchymal cell lines, which is in accord with E-cadherin expression in these cell lines.2. MiR-101overexpression significantly increased E-cadherin protein levels, while it downregulated mesenchymal markers fibronectin, N-cadherin, and vimentin. Ectopic miR-101expression significantly decreased cell migration and invasion.3. MiR-101could regulate the expression of ZEB1and ZEB2by directly targeting the3’-UTR of ZEB1or ZEB2.4. MiR-101could upregulate the expression of E-cadherin and inhibit EMT by directly targeting the3’-UTR of ZEB1or ZEB2, which is independent of miR-200family.