TCF21 Regulating KISS1 Inhibits The Proliferation、 Migration And Invasion Of Colorectal Cancer Cells Via Wnt/β-catenin Signaling Pathway

Objective:To study the mechanism of TCF21 inhibiting the proliferation,invasion and migration of colorectal cancer cells through KISS1 and Wnt/ cell-catenin signaling pathways.Methods:1.The m RNA and protein expression level of TCF21 and KISS1 in colorectal cancer and para-carcinoma normal tissues were detected by real-time fluorescence quantitative PCR and Immunohistochemical.2.Four colorectal cancer cell lines(SW480,LOVO,SW620,HCT116)were screened by real-time fluorescence quantitative PCR.The cell lines with the lowest TCF21 expression were selected to transfect TCF21 overexpressing lentivirus to construct overexpressing cell lines,and the cell lines with the highest TCF21 expression to transfect TCF21 interfering lentivirus to construct knockout cell lines.Then we divided the cell lines into blank control group(CON),overexpression group(TCF21),knockout group(sh TCF21)and empty vector group(NC)after the stable expression of TCF21 in each group.3.Western blot was demanded to measure the protein expression levels of TCF21,KISS1 and WNT3 a,β-catenin and N-cadherin.CCK-8 test was demanded to measure cell proliferation in each group.Cell scratch test was adopted to revealed the cell migration ability.At the last,the Transwell chamber detected the invasion capacity of cells in each group.4.The overexpression of KISS1 lentivirus was transfected into the TCF21 knockout group to detect the expression levels of TCF21,KISS1 and the proteins associated with the Wnt/ he-catenin signaling pathway,including WNT3 a,he-catenin and n-cadherin,as well as their proliferation,migration and invasion capacities.Results:1.In colorectal cancer,protein expression levels and m RNA expression levels of TCF21 and KISS1 were significantly lower than that of para-cancer normal tissues(P<0.001).2.The results of RT-q PCR and western blot showed that TCF21 expression was the lowest in HCT116 and the highest in SW480 among the four cell lines(SW480,LOVO,SW620 and HCT116).After TCF21 overexpressing in HCT116 cells,the TCF21 m RNA and protein expression levels were significantly increased(P<0.001).After nocking out TCF21 in SW480 cells,the TCF21 m RNA and protein expression levels were remarkably decreased(P<0.001).The overexpression group and the knockout group were stably constructed.3.In western blot experiment,compared with the control group,the KISS1 expression level was remarkabley increased in the TCF21 overexpression group(p<0.001),and the expression of WNT3 a,N-cadherin and β-catenin were decreased remarkably(p<0.001).In TCF21 knockout group,the KISS1 expression level was decreased remarkably(P<0.001),the expression level of WNT3 a,N-cadherin andβ-catenin were remarkably increased(P<0.001).After overexpressing KISS1 in the TCF21 knockout group,compared with TCF21 knockout group,the KISS1 expression level increased and the expressions of WNT3 a,N-cadherin and β-catenin decreased(p<0.001).4.In the cck-8 cell proliferation assay,compared with the control group,the OD values of the TCF21 overexpression group at 24 h,48h and 72 h were significantly reduced(p<0.05),indicating that the cell proliferation activity was weakened.In the TCF21 knockout group,OD values were significantly increased at 24 h,48h and 72h(p<0.05),indicating increased cell proliferation activity.The cell proliferation activity was partially reversed after overexpression of KISS1 in the TCF21 knockout group(p<0.05).5.In the cell scratch test,compared with the control group,the migration of cells in the TCF21 overexpression group was significantly reduced after 24h(P<0.05).The mobility of TCF21 knockout group increased significantly(p<0.01).After overexpression of KISS1 in TCF21 knockout group,cell mobility was decreased(p<0.05).6.Compared with the control group,the number of cells penetrating into the lower compartment was significantly reduced in the TCF21 overexpression group(p<0.001).The number of cells entering the lower compartment increased significantly in the TCF21 knockout group(p<0.001).After overexpression of KISS1 in the TCF21 knockout group,the number of penetrations into the lower compartment decreased(p<0.001).Conclusion:1.The expression of TCF21 and KISS1 in colorectal cancer tissues decreased,and TCF21 and KISS1 expression were positively correlated.2.TCF21 may effectively inhibit the metastatic ability of colorectal cancer cells by activating the KISS1 expression and inhibiting the Wnt/ β-catenin signaling pathway.