SNAI2 Functions As A CeRNA To Promote Invasion And Metastasis Of Ovarian Cancer Cells By Inducing MARCKS Expression

Part ⅠThe relationship between SNAI2 expression and the clinicopathological parameters in serous ovarian cancer patientsObjectives:To investigate the relationship between SNAI2 expression and the clinicopathological parameters in ovarian cancer patientsMethods:First, we analyzed the relationship between the SNAI2 expression and the clinicopathological features for 208 patients with serous ovarian cancer using data from the TCGA data portal.χ2 test was used to determine the statistical significances between different groups. Next, the Log-rank test was used to determine the relationship between SNAI2 expression and overall survival of ovarian cancer patients from TCGA dataset and GEO dataset(GSE26712). The Kaplan-Meier method was used to generate survival curves.Results:The results revealed that increased expression of SNAI2 was significantly correlated with FIGO stage (P=0.015) and lymphatic invasion status (P=0.004), whereas not with age(P>0.05) and histological grade(P>0.05). Patients with higher SNAI2 expression had a shorter OS (P=0.044, HR=0.65) in the TCGA dataset. This finding was further confirmed in another GEO dataset. Namely, higher expression of SNAI2 predicted poorer OS (P=0.0017, HR=0.56) in GSE26712 dataset.Conclusions:Increase expression of SNAI2 predicted a worse clinical outcome in patients with serous ovarian cancer.Part IIThe screen and identification of SNAI2 associated ceRNAsObjectives:To screen and identify SNAI2 associated ceRNAs.Methods:First, bioinformatics analysis was performed to screen SNAI2 associated ceRNAs. Next, Real-time PCR and Western Blot were employed to determine the regulatory effect of SNAI2-3’UTR on the expression of putative SNAI2 associated ceRNAs. Subsequently, correlation analysis was performed between SNAI2 and its related ceRNAs in ovarian cancer tissues from TCGA dataset and GSE26712 dataset. Next, Real-time PCR was used to determine the relative expression levels of miR-200c, miR-204, miR-211, miR-218 and miR-429 in OVCA433 and SKOV-3 cells. Dual luciferase reporter assay was employed to explore the regulatory effect of candidate miRNAs on MARCKS expression, and to determine whether SNAI2-3’UTR could induce MARCKS expression by functioning as a ceRNA.Results:Ten genes, including CDH2, FN1, KLF8, MARCKS, MMP2, PPARG, TWIST1, VIM, ZEB1 and ZEB2, were selected as putative SNAI2 associated ceRNAs. Among them, MARCKS was identified as a potential candidate of SNAI2 associated ceRNAs. Overexpresssion of SNAI2-3’UTR significantly induced MARCKS expression at both mRNA and protein level. Moreover, the expression of SNAI2 was positively correlated with the expression of MARCKS in ovarian cancer tissues from both TCGA dataset and GSE26712 dataset. miRNA profile analysis revealed that the expression level of miR-218, miR-200c and miR-429 were relatively higher than that of miR-204 and miR-211. Dual luciferase reporter assay revealed that miR-218 and miR-200c could significantly inhibit the luciferase activity by binding to MARCKS-3’UTR in a dose dependent manner, while miR-429 only had a modest regulatory effect on the luciferase activity. Moreover, SNAI2-3’UTR could rescue the inhibitory effect of miR-218 and miR-200c on the luciferase activity in a dose dependent manner.Conclusions:SNAI2 could function as a ceRNA to regulate MARCKS expression in ovarian cancer at least partly by competing for miR-218 and miR-200c.Part IIIExploration of the role of MARCKS in EMT process of ovarian cancer Objectives:To explore the role of MARCKS in EMT process of ovarian cancerMethods:Lentivirus mediated shRNA was used to generate cell lines that stably inhibited MARCKS expression. The knockdown efficiency was determined by Real-time PCR and Western Blot. The phenotypic changes resulted from MARCKS downregulation was monitored by RTCA-CIM-Plate. Moreover, we also determined the morphologic changes of ovarian cancer cells induced by decreased MARCKS expression.Results:MARCKS was significantly decreased in both OVCA433 and SKOV-3 cells after transfection with MARCKS shRNA. Subsequent functional studies revealed that MARCKS downregulation repressed invasion and migration of both OVCA433 and SKOV-3 cells and the cellular morphology become more like that of epithelial cells.。Conclusions:The EMT process driven by SNAI2 is mediated, at least partly, by inducing MARCKS expression through the ceRNA working model.Part IVExploration of the role of SNAI2-3’UTR in the invasion and metastasis of ovarian cancerObjectives:To investigate the role of SNAI2-3’UTR in the invasion and metastasis of ovarian cancerMethods:Gain-of-functional studies were performed in both OVCA433 and SKOV-3 cells. First, stable SNAI2-3’UTR-overexpressing cell lines were generated using lentivirus vector. Subsequent, RTCA-CIM-Plate was used to monitor the invasive and migratory abilities of ovarian cancer cells.Results:SNAI2-3’UTR overexpression significantly induced invasion of both OVCA433 and SKOV-3 cells, without significantly affecting their migratory abilities.Conclusions:SNAI2-3’UTR could promote the invasion of ovarian cancer cells.