RNA干扰沉默MIF基因对HeLa细胞生长、迁移和黏附性的影响  

Effects of silencing migration inhibitory factor gene by RNA interference on proliferation,invasion,and adhesion of HeLa cells

作　　者：戴波[1,2] 肖定璋[1] 余细勇[1] 郭爱林[1] 谢至[1] 

机构地区：[1]广东省人民医院医学研究中心,广州510080 [2]南方医科大学基础医学院,广州510515

出　　处：《肿瘤》2009年第5期413-418,共6页Tumor

基　　金：广东省自然科学基金资助项目(编号:06020897)

摘　　要：目的:探讨巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)对宫颈癌HeLa细胞生长、迁移和黏附能力的影响。方法:通过反转录病毒载体介导RNA干扰,获得稳定抑制MIF表达的HeLa细胞株。Western印迹法检测MIF蛋白沉默效果以及FAK、ICAM-1、Bax和Bcl-2蛋白表达情况。观察HeLa细胞生长状态的变化,采用迁移实验、黏附实验和软琼脂糖克隆形成实验检测MIF对HeLa细胞迁移和黏附能力的影响。通过裸鼠成瘤实验观察沉默MIF基因后HeLa细胞的成瘤效果。肿瘤坏死因子(tumor necrosis factor α,TNF-α)和放线菌酮(cycloheximide,CHX)诱导细胞凋亡后,FCM法检测沉默MIF基因对HeLa细胞凋亡的影响。结果:构建获得稳定抑制MIF蛋白表达的HeLa-pSUPER-MIF细胞株。MIF基因沉默后细胞生长减慢,迁移能力和黏附功能减弱;HeLa细胞中FAK、ICAM-1的表达量减少,无法在裸鼠中形成肿瘤;同时能减少TNF-α和CHX诱导的细胞凋亡,并且使Bax表达量下降。结论:MIF通过影响HeLa细胞中FAK、ICAM-1的表达,促进肿瘤细胞生长,增加肿瘤细胞的迁移和黏附能力,因此MIF可能是肿瘤形成过程中的重要因子。[ABSTRACT]Objective：To investigate the effect of macrophage migration inhibitory factor （MIF）on proliferation, invasion, and adhesion of human cervical HeLa cells. Methods： MIF gene was silenced by small interfering RNA （siRNA） via retrovirus mediation and obtained a stable human cervical cancer HeLa cell line with silencing of MIF gene. Western blotting was used to detect the silencing efficacy of MIF protein and the expression of FAK, ICAM-1, Bax and Bcl-2 protein.The growth of HeLa cells was observed. The motility and adhesion capacity of HeLa cells were determined by using migration assay, adhesion assay, and soft-agar colony formation test. In vivo,tumorigenesis capacity of MIF gene-silenced HeLa cells was observed in nude mice. The effect of silencing MIF gene on apoptosis of HeLa cells induced by tumor necrosis factor α（TNF-α） and cycloheximide （CHX） were detected by flow cytometry. Results： HeLa-pSUPER-MIF cell line was onstructed and obtained, in which the MIF protein expression was effectively knocking down.The HeLa-pSUPER-MIF cell line grew slowly and its motility and adhesion capacity were weakened. The expression of FAK and ICAM-1 was decreased in HeLa-pSUPER MIF cell line.Tumor formation of HeLa-pSUPER-MIF cells was not observed in nude mice. Silencing MIF gene inhibited the apoptosis of HeLa cells induced by TNF-α and CHX and induced decrease in Bax expression. Conclusion：MIF can promote the proliferation of HeLa cells, keep the motility of tumor cells, and increase the adhesion capacity by regulating the expression of FAK and ICAM-1.MIF may be one of the important factors in tumorigenesis.

关 键 词：宫颈肿瘤 RNA干扰 巨噬细胞游走抑制因子 细胞生理学 HELA细胞 

分 类 号：R737.33[医药卫生—肿瘤] R730.231[医药卫生—临床医学]