shRNA沉默survivin基因对人膀胱移行细胞癌T24细胞的生物学行为的影响.docVIP

PAGE PAGE 2 shRNA 抑制沉默 survivin 基因对人膀胱移行细胞癌T24细胞的生物学行为的影响 杜 虎1 ,吴小候1 ,罗春丽2 ,刘 川1 ,曾 柯1 ,何云锋1 ,尹志康1（重庆医科大学:1：1附属第一医院泌尿外科，2检验系，重庆 400016） 摘 要：目的 构建survivin特异性短发夹RNA（shRNA）的表达载体，探讨其对人膀胱移行细胞癌T24细胞的生物学行为的影响。方法 将构建survivin特异shRNA表达载体转染T24细胞，RT-PCR、Western blot检测survivin 的mRNA及其蛋白的表达水平；通过细胞生长曲线、随机运动实验、Matrigel穿膜实验检测肿瘤细胞生长、运动及体外侵袭能力。结果 经EcoR Ⅰ和 Hind IIIⅢ双酶切和测序鉴定，成功筛选含目的 DNA 片段的重组载体pshRNA-survivn2，并能够成功转染T24细胞；转染pshRNA-survivn2后可显著抑制细胞中survivin 的mRNA及其蛋白的表达，抑制率分别为61.73%和73.27%；pshRNA-survivin2组的细胞侵袭力与运动能力均有明显的下降，第3天、第5天时肿瘤细胞生长抑制率分别为59.13%、83.86%。结论 应用PTZU6+1质粒载体构建survivin的shRNA表达载体，成功转染T24细胞，有效抑制其survivin 的mRNA及其蛋白的表达，并可抑制T24细胞的增殖、运动及体外侵袭能力。 关键词：survivin；short hairpin RNA；肿瘤侵袭；膀胱肿瘤 中图法分类号： 文献标识码：A Inhibition of endogenous survivin gene expression in T24 bladder carcinoma cells by shRNA DU Hu，WU Xiao-hou，LUO Chun-li，LIU Chuan，ZENG Ke，HE Yunfeng，YIN Zhi-kang（Department of Urological Surgery, The First Affiliated Hospital, Chongqing University of Medical Science, Chongqing 400016, China） Abstract:Objective To construct the plasmid containing short hairpin RNA（shRNA）of survivin，and to suppress the expression of endogenous survivin gene in T24 cells. Methods The recombinant plasmid pshRNA-survivin2 expression construct was confirmed by using EcoR I and Hind III double digestion and by sequencing. The plasmid pshRNA-survivin2 was stably transfected into TCCB cells T24 to detect effect of survivin expression and analyze the inhibition of survivin gene with semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) and western blot. The biological effects were observed, including anchorage-independent growth, in vitro invasion by cell migration assay and tranwell chamber assay. Results The expression of survivin in transfected T24 cells was markedly depressed at both mRNA and protein levels(61.73%,73.37%,respectively) as compared with control. The growth of tumor cells was retared by anchorage-independent growth assay. The inhibitory percentages of tumor cell