短发夹RNA靶向沉默叉头框蛋白Q1基因对膀胱癌上皮间质转化的逆转作用  

Reversal effect of short hairpin RNA targeting forkhead box protein Q1 gene silencing on epithelial- mesenchymal transition in bladder cancer

作　　者：朱智能[1] 朱朝辉[1] 庞自力[1] 兰东阳[1] 王海鹏[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院泌尿外科,武汉430022

出　　处：《中华实验外科杂志》2013年第4期765-768,F0003,共5页Chinese Journal of Experimental Surgery

摘　　要：目的观察转录因子叉头框蛋白Ql（FOXQl）基因沉默对膀胱癌T24细胞上皮间质转化（EMT）的逆转，探讨其促进肿瘤侵袭转移的作用机制。方法构建针对FOXQl基因的短发卡RNA（shRNA）真核表达载体，脂质体Lipofactamine2000介导FOXQl干扰质粒转染人膀胱癌细胞株T24细胞；转染48h后采用逆转录-聚合酶链反应（RT—PCR）、Westernblot法检测FOXQl、上皮间质标志物E-钙黏蛋白（E-Cadherin）、N-钙黏蛋白（N—Cadherin）和波形蛋白（Vimentin）的表达改变；细胞划痕实验、Transwell小室侵袭实验检测体外细胞迁移和侵袭能力；噻唑蓝（M1Tr）比色法和吖啶橙／溴乙锭（AO／EB）荧光染色法检测shRNA对细胞增殖和凋亡的作用。结果与转染NC—shRNA阴性对照组比较，转染FOXQl-shRNA48h后的T24细胞，FOXQl和间质标记基因N—Cadherin、Vimentin的mRNA或蛋白表达下降，而上皮标记基因E．cadherin的mRNA或蛋白表达显著增加；转染后的T24细胞形态向正常上皮细胞转化，体外迁移能力与侵袭力下降[（15．4±1．4）％比（76．5±1．1）％，P〈0．05]；细胞增殖明显抑制（57．8％比82．7％，P〈0．05），同时细胞凋亡率增加（37．6％比14．2％，P〈0．05）。结论靶向沉默FOXQl基因可以逆转肿瘤细胞的间质表型向正常上皮表型转化，抑制癌细胞迁移和侵袭，降低肿瘤转移潜能。Objective To investigate the reversal effect of epithelial-mesenehymal transition （EMT） by silencing transcription factor forkhead box protein Q1 （FOXQ1） in human bladder cancer cell line T24, and study its role in invasion and metastasis of T24 cells. Methods Short hairpin RNA （shRNA） eukaryotic expression vector （FOXQI-shRNA） targeting human FOXQ1 gene was transfeeted in- to T24 cells with high metastatic potential by lipofeetamine 2000. The expression of FOXQ1 and epithelial mesenchymal markers （ E-Cadherin, N-Cadherin, vimentin） was detected by using reverse tmnseription- polymerase chain reaction （RT-PCR） and Western blotting after transfectian for 48 h. The migration and metastatic potentials of T24 cells were examined by cell wound model and Transwell chamber assay in vitro respeetively, and the proliferation of T24 cells was determined by using MTT assay. Acridine orange/ethid- ium bromide （AO/EB） fluorescent staining was performed to detect apoptosis. Results In the FOXQ1- shRNA transfection group, the expression of FOXQ1, N-Cadherin and Vimenfin was decreased in the T24 cells remarkably, and the expression of E-Cadherin was significantly up-regulated （ P 〈 0. 05 ）. The mor- phology of T24 cells was transformed into a normal epithelial phenotype, and the motility and invasion of T24 ceils [ （ 15.4 ± 1.4） % vs （76. 5 ± 1.1 ） %, P 〈 0.05 ] were inhibited, the abilities of proliferation were inhibited significantly （ 57.8% vs 82. 7% , P 〈 O. 05 ） in vitro, and apoptosis rate （ 37. 6% vs 14. 2% ,P 〈 0. 05 ） was increased notablely as compared with the negative control group （NC-shRNA）. Conclusion The silencing of FOXQ1 may reverse mesenchymal morphology into a normal epithelial phe- notype, inhibit cancer cell migration and invasion, and suppress tumor metastatic potential.

关 键 词：上皮间质转化 转录因子叉头框蛋白Q1 膀胱癌 RNA干扰 

分 类 号：R737[医药卫生—肿瘤]