过表达FOXF2对人宫颈癌SiHa细胞迁移及增殖影响机制探讨

Mechanism of overexpression of FOXF2 on the migration and proliferation of human cervical cancer SiHa cells

目的探讨过表达叉头框转录因子F2(FOXF2)调控HPV早期基因区6(early region,E6)和早期基因区7(early region,E7)抑制人宫颈癌SiHa细胞迁移、增殖的影响及其作用机制。方法构建慢病毒转染SiHa细胞,实验分为对照组和实验组,qRT-PCR和蛋白质印迹法检测过表达慢病毒对照组及实验组中FOXF2、E6、E7、p53、pRB/RB、E2F1mRNA和(或)蛋白表达变化;划痕试验和平板克隆形成试验分别检测细胞迁移和增殖能力。SPSS 20.0对数据进行统计学分析。结果 SiHa细胞内FOXF2过表达后,过表达慢病毒实验组FOXF2mRNA表达水平为20.07±0.63,高于对照组的1.05±0.08,差异有统计学意义,F=272.883,P<0.001;实验组p53mRNA表达水平为1.46±0.07,高于对照组的0.95±0.16,差异有统计学意义,F=25.775,P=0.007;实验组E2F1mRNA表达水平为0.77±0.06,低于对照组的1.00±0.02,差异有统计学意义,F=39.74,P=0.003;实验组RB mRNA表达水平为1.22±0.09,高于对照组的1.00±0.04,差异有统计学意义,F=14.738,P=0.018。过表达慢病毒实验组FOXF2蛋白表达水平为0.41±0.05,高于对照组的0.21±0.06,差异有统计学意义,F=34.192,P<0.001;RB蛋白表达水平为0.68±0.06,高于对照组的0.40±0.03,差异有统计学意义,F=71.468,P=0.001;pRB蛋白表达水平为0.45±0.06,低于对照组的0.75±0.09,差异有统计学意义,F=25.480,P=0.007;E2F1蛋白表达水平为0.32±0.03,低于对照组的0.57±0.06,差异有统计学意义,F=43.872,P=0.003;p53蛋白表达水平为0.22±0.03,高于与对照组的0.11±0.01,差异有统计学意义,F=36.327,P=0.004;E6蛋白表达水平为0.21±0.03,低于对照组的0.40±0.09,差异有统计学意义,F=12.36,P=0.025;E7蛋白表达水平为0.21±0.03,低于对照组的0.36±0.08,差异有统计学意义,F=9.093,P=0.039。实验组细胞迁移率为(0.36±0.06)%,低于对照组的(0.74±0.06)%,差异有统计学意义,F=60.945,P=0.001。实验组细胞克隆形成率为(0.34±0.04)%,低于对照组的(0.67±0.07)%,差异有统计学意义,F=49.751,P=0.002。结论 FOXF2通过调控HPV E6/E7而抑制SiHa细胞的体外迁移及细胞增殖能力,促进p53的表达,抑制pRB及E2F1表达。FOXF2可能通过下调HPV E6/E7抑制宫颈癌的发生发展。

Objective This study aimed to study the effect and mechanism of overexpression of forkhead box F2(FOXF2)regulate HPV E6/E7 inhibiting the migration and proliferation behavior in SiHa cells.Methods The expressions of FOXF2,E6,E7,p53,pRB and E2 F1 mRNA or protein in SiHa treated with LV5-NC or LV5-FOXF2 were detected by qRT-PCR and Western blot.Wound healing assay and colony formation in vitro were performed to detect the migration and proliferation behavior in SiHa treated with LV5-NC or LV5-FOXF2.Results The expression of FOXF2 mRNA in the experience group(20.07±0.63)was higher than the control group(1.05±0.08,F=272.883,P<0.001).The expression of p53 mRNA in the experience group(1.46±0.07)was higher than the control group(0.95±0.16,F=25.775,P=0.007).The expression of E2 F1 mRNA in the experience group(0.77±0.06)was lower than the control group(1.00±0.02,F=39.743,P=0.003).The expression of RB mRNA in the experience group(1.22±0.09)was higher than the control group(1.00±0.04,F=14.738,P=0.018).The results of western blot demonstrated that the expressions of FOXF2 protein in the experience group(0.41±0.05)was higher than the control group(0.21±0.06,F=34.192,P<0.001).The expressions of RB protein in the experience group(0.68±0.06)was higher than the control group(0.40±0.03,F=71.468,P=0.001).The expressions of pRB protein in the experience group(0.45±0.06)was lower than the control group(0.75±0.09,F=25.480,P=0.007).The expressions of E2 F1 protein in the experience group(0.32±0.03)was lower than the control group(0.57±0.06,F=43.872,P=0.003).The expressions of p53 protein in the experience group(0.22±0.03)was higher than the control group(0.11±0.01,F=36.327,P=0.004).The expressions of E6 protein in the experience group(0.21±0.03)was lower than the control group(0.40±0.09,F=12.360,P=0.025).The expressions of E7 protein in the experience group(0.21±0.03)was lower than the control group(0.36±0.08,F=9.093,P=0.039).By wound healing assay the ability of migration in the experience group[(0.36±0.06)%]was lower than the control group(0.74±0.06)%,F=60.945,P=0.001.By colony formation,the ability of proliferation in the experience group[(0.34±0.04)%]was lower than the control group(0.67±0.07)%,F=49.75,P=0.002.Conclusion Overexpression of FOXF2 resulted in the expression of E6,E7,pRB,E2 F1 inhibition and p53 increase,cell proliferation weaken,and cell migration behavior inhibition in SiHa cells,which in turn inhibited the occurrence and development of cervical cancer.