小分子干扰RNA干扰RhoGDI2基因表达对结肠癌细胞凋亡迁移的影响及其机制  

Effects of RhoGDI2 gene silencing by siRNA on apoptosis and invasion of colon carcinoma cell lines

作　　者：李守峰[1] 刘璐[1] 伍衡[1] 许鹤洋[1] 来伟[1] 褚忠华[1] 

机构地区：[1]中山大学孙逸仙纪念医院胃肠外科,广州510120

出　　处：《中华实验外科杂志》2012年第10期2000-2002,共3页Chinese Journal of Experimental Surgery

基　　金：广东省自然科学基金自由申请项目（10151008901000071）

摘　　要：目的探讨小分子干扰RNA（siRNA）干扰RhoGD12基因的表达对结肠癌细胞株凋亡侵袭的影响及其机制。方法运用Westernblot和实时定量逆转录聚合酶链反应（RT—qPCR）检测结肠癌细胞株LoVo、SW480、CaC02、DLD1、RKO、HT-29的RhoGD12表达。设计及合成RhoGD12 siRNA干扰序列，通过Lipofectamine“2000转染到筛选细胞，细胞分为干扰组和空白对照、阴性对照组；膜联蛋白V／碘化丙锭（Annexin V／PI）双染流式细胞仪检测细胞凋亡率，细胞划痕试验和Transwell实验检测细胞迁移能力，Westemblot检测上皮问质转化（EMT）相关E一钙黏蛋白（E—cadherin）／波形蛋白（Vimentin）、Snail转录因子表达水平。结果野生型人结肠癌细胞株RhoGDl2表达量由高到低依次是DLD1、HT29、RKO、CaC02、LoVo、SW480；LoVo细胞siRNA干扰后RhoGD12表达抑制率大于70％；干扰组、阴性对照组、空白对照组细胞凋亡率分别是（2．67±0．97）％、（2．45±0．74）％、（2．35±0．43）％（P〉0．05）；干扰组及对照组的细胞划痕24h愈合度分别是90％、50％（P〈0．05）；Tran．swell实验24h迁移细胞数目：干扰组为55．7±9．5，阴性对照组和空白对照组分别为25．1±8．6、32．3±12．9（P〈0．05）；siRNA后结肠癌细胞E—cadherin蛋白表达下调，Vimentin蛋白、Snail转录因子表达上调。结论结肠癌RhoGD12基因沉默可能通过引起EMT促进细胞迁移。Objective To investigate the effect of the silencing RhoGDI2 gene expression by small interfering RNA （siRNA） on the apoptosis and invasion of colon cancer cell lines. Methods With Western blotting analysis and real-time reverse transcription-polymerase chain reaction （RT-qPCR）, RhoGDI2 expression of the colon cancer cell lines of LoVo, SW480, CaCo2, DLD1, RKO, the HT-29 was detected. The siRNA of RhoGDI2 with LipofectamineTM2000 was transfected into target cells, comprising siRNA and control groups ; Annexin V/PI staining flow cytometry to detect apoptosis, with Wound healing assay and the Transwell plate migration and invasion was detected. The epithelial-mesenchymal transition （EMT） relevant protein E-cadherin/Vimentin and Snail expression was detected. Results Human colon cancer cell lines RhoGDL2 expression levels decreased in the order of DLD1, of HT29, RKO, CaCo2, LoVo, SW480 ; siRNA inhibited RhoGDI2 expression rate of LoVo cell by 70% ;in silence group, negative control group and blank control group, the apoptosis rates were （2. 67 ＋ 0. 97） % （2.45 ~ 0. 74） % （2. 35 ~ 0. 43 ） % （ P 〉 0. 05 ） ; Wound healing assay and Transwell assay suggested RhoGDI2 silencing could improve migration ; siRNA interference of colon cancer cells downregulatd E-cadherin, but upregulated Vimentin protein of Snail expression. Conclusion siRhoGDI2 could significantly facilitate the cell proliferation, migration, invasion of colon cell.

关 键 词：小分子干扰RNA RhoGDI2 结肠癌 上皮间质转化 细胞迁移 

分 类 号：R735.35[医药卫生—肿瘤]