sirna干扰foxf2对人宫颈癌siha细胞体外转移的影响.docVIP

siRNA干扰FOXF2对人宫颈癌SiHa细胞体外转移的影响 　　[摘要]目的 研究siRNA干扰叉头框转录因子F2（FOXF2）对人宫颈癌SiHa细胞体外转移能力的影响及其作用机制。方法 采用RT-PCR、Western blot分别检测siRNA-FOXF2转染前后，SiHa细胞中FOXF2 mRNA、蛋白的表达变化；同时用划痕试验和Transwell侵袭试验分别检测siRNA-FOXF2转染前后细胞的迁移和侵袭能力，CCK8检测siRNA-FOXF2转染前后细胞的增殖能力。结果 SiHa细胞内FOXF2敲除后，RT-PCR、WB检测显示siRNA-FOXF2转染组FOXF2的蛋白、mRNA均明显下降；划痕试验和Transwell侵袭试验显示细胞侵袭、迁移能力增强；CCK8检测显示siRNA-FOXF2转染组促进细胞增殖能力（P 　　[关键词]宫颈癌；叉头框转录因子F2；SiHa细胞；细胞迁移；细胞侵袭；细胞增殖 　　[中图分类号] R737.3 [文献标识码] A [文章编号] 1674-4721（2017）03（c）-0004-04 　　[Abstract]Objective To explore the effect of siRNA silencing FOXF2 on invitro metastasis of human cervical carcinoma SiHa cells and its mechanism.Methods The expression of FOXF2 mRNA and protein in SiHa-siRNA-FOXF2 cells and negative control cells were detected by RT-PCR and Western blot respectively. The migration and invasion ability of cells were detected by wound-healing assay and Transwell assay before and after siRNA-FOXF2 transfection.CCK8 was used to detect the proliferation of cells before and after siRNA-FOXF2 transfection.Results The expression of FOXF2 mRNA and protein in siRNA-FOXF2 transfected group was significantly decreased after FOXF2 knockout in SiHa cells by RT-PCR and WB detection；the invasion and migration were enhanced by wound-healing assay and transwell assay.After transfection of siRNA-FOXF2 into cervical cancer SiHa cell lines，the capability of cell proliferation was significantly enhanced （P 　　1.2方法 　　1.2.1细胞培养 SiHa细胞复苏后，加入含10%FBS、100 U/ml青霉素及100 μg/ml链霉素的DEME中培养，置入37℃、5%CO2培养箱内，24 h内换液。取对数生长期的细胞，用0.25%胰酶消化、传代、冻存。 　　1.2.2细胞转染 将培养SiHa细胞分为5组：转染阴性对照组（阴性）、Si-FOXF2 1415组（1415）、Si-FOXF2 1305组（1305）、Si-FOXF2 650组（650）及未经任何处理的SiHa细胞作为对照组（NC）。转染前一天将SiHa细胞接种于6孔板中，转染时细胞融合达到30%～50%，PBS清洗3遍，每孔加入900 μl无血清培养基。将5 μl的siRNA和3 μl的Lipo3000分别与50 μl的OptiMEMⅠMedium混悬，轻轻混匀后静置5 min。将含有siRNA和Lipo3000的OptiMEMⅠ Medium轻轻混匀后静置20 min。将每孔100 μl混匀的DNA脂质体复合物均匀滴入含有无血清培养基的细胞中。48 h后终止提样。 　　1.2.3 RT-PCR检测各组FOXF2 mRNA的表达 终点提取RNA，采用紫外/可见光分光光度仪测量细胞样品总RNA的浓度和纯度[D（260 nm）/D（280 nm）分析RNA浓度在1.9～2.1之间提示RN