The Role Of Cytoskeleton Regulator Protein Mena In ESCC Cell Proliferation,migration,invasion And Its Underlying Molecular Mechanisms

Background and ObjectiveEsophageal cancer is the one of the most frequently occurring cancer worldwide,harboring high incidence in East Asian coutries[2].EC is divided into two main histological types:esophageal adenocarcinoma(EAC)which is more common in the developed countries,such as North America and Europe[3,4],and esophageal squamous cell carcinoma(ESCC)that mainly occurring in Asia[5,6],especially in China[3,4].At present,ESCC is still the main histological subtype,accounting for more than 90%of cases of all esophageal cancers,and 5-year survival rate of the patients with ESCC remains quite poor[7,8].Most importantly,about 50%of the patients with ESCC exhibit the local invasion and metastasis at the time of diagnosis,which will lead to the high mortality[9,10]Despite recent advances in therapeutic strategies,a large number of patients with ESCC remain poor clinical outcome.Therefore,it is particularly necessary to seek the novel molecular target for therapy of the patients with ESCC.Human Mena(hMENA)encoded by the ENAH gene,is a member of the Ena/VASP family of actin filament elongation factors[11]Recently,several researches found Mena was up-regulated in many human cancers[14].In addition,Mena is correlated with tumor size,advanced clinical stages and highly invasive properties in breast cancer[15,16].However,no report about Mena expression and its biological function in ESCC cell have not been explored.In this study,we analyzed Mena gene expression levels in ESCC samples and examined Mena expression in a panel of ESCC cell furthermore We investigated the effects of Mena knockdown on tumor cell migration and invasion as well as the capability of cell proliferation,and further preliminarily elucidated the possible molecular mechanisms.We performed several experiments to explore the functional role of Mena in ESCC.First,we examined mRNA levels by quantitative real-time quantitative PCR(qRT-PCR)in 27 human primary ESCC tumor tissue and matched adjacent normal tissues.Second,we detected Mena protein expression level among ESCC cell lines and normal esophageal epithelial cell,we selected TE13 cells which yield the highest MENA expression and TE1 which showed the lowest Mena expression,then we compared cell proliferation,colony formation,migration and invasion between TE1 cells and TE13 cells.To further investigate whether Mena could promote ESCC cell invasion and migration,we constructed siRNA for knock down Mena expression and MENA expression vector for elevate Mena expression,we explored ESCC cell proliferation,colony formation,migration and invasion after interfering Mena expression.To explore the underlying mechanism,first,we detected the expression of EMT specific marker,Matrix metalloproteinases and the Akt signaling pathway between Mena interfered group and control.Methods1 Mena expression pattern in ESCC patient.Collecting ESCC primary tumor tissue and matched adjacent normal tissues from the first affiliated hospital of Zhengzhou University,extract total mRNA of tumor tissue and matched adjacent normal tissues,quantitative Real-Time PCR detect Mena mRNA expression level of tumor tissue and adjacent normal tissue.2 Comparison of biological behavior between ESCC cells with different Mena expression level.First,we detected Mena protein expression level between ESCC cell lines and normal esophageal epithelial cell via western blot,than selected TE13 cell with the highest Mena expression level and TE1 cell with the lowest for detection of biological behavior.We detected cell proliferation,colony formation,cell migration and invasion respectively via CCK-8 assay,colony formation assay,wound-healing assay and transwell assay between ESCC cells with different Mena expression level.3 siRNA knock down Mena expression in TE13 cells.To further explore function of Mena in ESCC cells,we knock down Mena expression by transfected Mena-siRNA into TE13 cells,and we detected cell proliferation,colony formation,cell migration and invasion respectively via CCK-8 assay,colony formation assay,wound-healing assay and Transwell assay between siNC group and siMena group.Than we detected EMT-specific marker(E-cadherin,N-cadherin,Snail,Slug),MMP-2,MMP-9,Akt and phosphorylated-Akt expression in siNC group and siMENA group repectively.4 Mena expression vector promotes Mena expression level in TE1 cells.We constructed Mena expression vector and transfected into TE1 cells to promotes Mena expression.we conducted CCK-8 assay,colony formation assay,wound-healing assay and Transwell assay to detect cell proliferation,colony formation,cell migration and invasion respectively between control group which is transfected with empty vector and Mena group transfected with Mena expression vector.We also detected EMT-specific marker(E-cadherin,N-cadherin,Snail,Slug),MMP-2,MMP-9,Akt and phosphorylated-Akt expression in Control group and MENA group.5 Statistical analysisStatistical analyses were carried out with the Statistical Package for the Social Sciences version 21.0(SPSS Inc.,Chicago,IL,USA).All data are expressed as mean± standard error(SEM)The comparisons of two samples were carried out using t test and paired-sample t test.The comparisons of two samples were carried out using t test and the comparisons of multiple samples were performed by one-way ANOVA..p<.05 was considered as statistical significance.ResultsThe Mena mRNA expression level was significantly higher in 22(81.5%)ESCC tissues compared with the adjacent non-tumor tissues(P<0.05).2 Biological behavior comparison between ESCC cells with different Mena expression level.Mena expression level among ESCC cells is higher than normal esophageal epithelial cell,proliferation,colony formation,migration and invasion of TE13 cells with the highest Mena expression level is significantly stronger than TE1 cells which expressed the lowest Mena protein.3.Knock down Mena expression in TE13 cellsExperiments showed that knock down Mena expression in TE13 cells suppressed cell proliferation(P<0.05),colony formation(P<0.05),migration(P<0.05)and invasion(P<0.05).Western blot analysis showed that knock Mena expression down regulated MMP-2(P<0.05),MMP-9(P<0.05),Akt(P<0.05)and p-Akt(P<0.05),meanwhile,EMT specific marker N-cadherin(P<0.05),Snail(P<0.05),and Slug(P<0.05)are also down regulated.4.Over expression of Mena in TE1 cellsOver expression Mena promoted cell proliferation,colony formation,migration and invasion.MMP-2(P<0.05),MMP-9(P<0.05),Akt(P<0.05)and p-Akt(P<0.05)expression is higher in TE1 cells transfected with Mena vector compared with TE1 cells transfected with empty vector.Western blot analysis of EMT specific marker showed that E-cadherin(P<0.05)expression is suppressed and N-cadherin(P<0.05),Snail(P<0.05)and Slug(P<0.05)were promoted in Mena group compared with Control group.Conclusions1 Mena expression level in ESCC tumor tissue and ESCC cell lines is higher than normal adjacent tissue and normal esophageal epithelial cell.2 Mena mediate ESCC cell proliferation,colony formation,invasion and migration via PI3K/Akt signaling pathway,affecting MMP-2 MMP-9 expression and EMT.