TMEFF1 Regulated By Transcription Factor P53 Promotes Malignancy Progression In Epithelial Ovarian Cancer Cell And The Mechanism Investigation

Objective:Based on the preliminary work,we first measured the expression of TMEFF1 in different epithelial ovarian cancer cell lines,and then constructed stable cell line models for over and inhibited expression of TMEFF1,respectively.The aim of this study was to explore the influence of TMEFF1 on the malignant biological behaviors in ovarian cancer cells,to identify activation pathways and regulation factors of TMEFF1,and to provide new ideas for ovarian cancer research.Methods:1.The expression of TMEFF1 in normal ovarian cell line and ovarian cancer cell lines was detected by Real-time PCR and Western blot.TMEFF1 stably overexpressing epithelial ovarian cancer cell lines OVCAR3-TMEFF1-H,ES-2-TMEFF1-H and their control cell lines OVCAR3-TMEFF1-H-Mock,ES-2-TMEFF1-HMock were constructed using plasmids.TMEFF1 stably inhibited expressing epithelial ovarian cancer cell lines CAOV3-TMEFF1-L,SKOV3-TMEFF1-L and their control cell lines CAOV3-TMEFF1-L-Mock and SKOV3-TMEFF1-L-Mock were constructed by lentiviral shRNA.The expression changes of TMEFF1 before and after transfection were detected by Real-time PCR,Western blot and immunocytochemistry.The proliferation ability of ovarian cancer cells was detected by MTT.The cell migration and invasion abilities were detected by scratch and Transwell assays.Apoptosis and cell cycle were detected by flow cytometry.2.Western blot was used to detect the expression of RAF,pRAF,MEK,p-MEK,ERK and p-ERK node proteins in the MAPK signaling pathway;expression of PI3 K,p-PI3 K,AKT,p-AKT node proteins in the PI3K/AKT signaling pathway;expression of EMT-related proteins E-cadherin,N-cadherin,Vimentin,MMP2,and MMP9 in TMEFF1 over and inhibited cell lines.Subsequently,MTT assay,scratch assay,Transwell assay and flow cytometry were used to detect the changes of proliferation,migration and invasion,apoptosis and cell cycle in epithelial ovarian cancer cells overexpressing TMEFF1 before and after the addition of pathway inhibitors.3.ChIP assay was used to detect if the transcription factor p53 can directly bind to the promoter region of TMEFF1 gene in ovarian cancer cell lines.Changes in the expression of TMEFF1 were detected by Western blot after the inhibition of TP53 gene expression.We investigated the correlation between TMEFF1 and AHNAK antigen with the aid of co-immunoprecipitation and double-label immunofluorescence analyses.Results: 1.The mRNA and protein expression levels of TMEFF1 in epithelial ovarian cancer cell lines CAOV3 and SKOV3 were higher than those in ES-2 and OVCAR3;however,they were lowest in normal ovarian epithelial cells(HOSEpiC).After overexpressing TMEFF1 in ovarian cancer cell line OVCAR3,the cell proliferation ability was increased,the migration and invasion abilities were increased,the apoptosis rate was decreased,the G0/G1 phase was shortened,and the S phase and G2/M phase were prolonged.After the inhibition of TMEFF1 expression in ovarian cancer cell line CAOV3,the cell proliferation ability was decreased,the migration and invasion abilities were decreased,the apoptosis rate was increased,the G0/G1 phase was prolonged,and the S phase and G2/M phase were shortened.2.The expression proportions of p-Raf/Raf,p-MEK/MEK,p-ERK/ERK,p-PI3K/PI3 K and p-AKT/AKT in cells increased after the overexpression of the TMEFF1 gene;but these decreased in CAOV3 cells after inhibition of the TMEFF1 gene.After overexpression of the TMEFF1 gene,the expression of an epithelial marker protein(E-cadherin)decreased,and that of mesenchymal marker proteins(vimentin and N-cadherin),as well as MMP2 and MMP,increased.After the inhibition of TMEFF1 gene expression,the expression of vimentin,N-cadherin,MMP2 and MMP9 decreased,but that of E-cadherin significantly increased.After the addition of MAPK or PI3 K pathway inhibitors,proliferation decreased significantly;the apoptosis rate increased significantly and the migration and invasion significantly decreased.3.After the inhibition of TP53 expression,TMEFF1 expression was significantly downregulated.As shown in ChIP assay,the transcription factor,p53,in CAOV3 cells bound to the promoter region of TMEFF1 and thus participated in the regulation of TMEFF1 expression.TMEFF1 and AHNAK interaction was then validated in ovarian cancer cell lines by co-immunoprecipitation.Confocal laser scanning microscopy showed the colocalization of the TMEFF1 and AHNAK in the ovarian cancer cell lines,which were both present in cell membrane and cytoplasm.Conclusion:1.TMEFF1 expression was increased in EOC.TMEFF1 promoted the proliferation,invasion and migration of ovarian cancer cells,and inhibited their apoptosis.2.TMEFF1 was involved in the malignant biological behaviors of tumor cells by activating PI3K/AKT and MAPK signaling pathways.3.TMEFF1 is a newly-found target gene of p53,and its expression is regulated through p53 transcription,and TMEFF1 interacts with the protein AHNAK.