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

Inhibitory effects of shRNA-silencing endogenous survivin gene on T24 bladder carcinoma cells

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 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EcoRⅠ和HindⅢ双酶切和测序鉴定,成功筛选含目的DNA片段的重组载体pshRNA-survivin2,并成功转染T24细胞;转染pshRNA-survivin2后可显著抑制细胞中survivin mRNA及其蛋白的表达,抑制率分别为61.73%和73.27%;pshRNA-sur-vivin2组的细胞侵袭力与运动能力均有明显的下降,第3、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,能有效抑制其survivin 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。

Objective To construct the plasmid containing short hairpin RNA （shRNA） of survivin to suppress the expression of endogenous survivin gene in T24 cells. Methods The recombinant plasmid pshR- NA-survivin2 expression construct was confirmed by EcoR Ⅰ and Hind Ⅲ double digestion and by sequencing. The plasmid pshRNA-survivin2 was stably transfected into transitional cell carcinoma of the bladder （TCCB） cells T24. Then survivin mRNA and protein expressions in the transfected T24 cells were detected by semi- quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. The biological effects of the plasmid pshRNA-survivin2 on T24 cells were evaluated through the detection of their anchorage-independent growth and in vitro invasion by cell migration assay and tranwcll chamber assay. Results The expression of survivin in transfected T24 cells was markedly depressed at both mRNA and protein levels （61.73% and 73.37%, respectively） as compared with control. Anchorage-independent growth assay showed the growth of tumor cells was retarded. The inhibitory percentage of transfected T24 cells was respectively 59. 13% and 83.86% on the third day and the fifth day. Transwell chamber assay showed the invasion ability of T24 cells was inhibited significantly. Conclusion The pshRNA-survivin2 expression plasmid, constructed from plasmid pTZU6 ＋ 1, can be successfully transfected into TCCB cell line T24 and can effectively inhibit the expression of survivin mRNA and protein. Therefore, the proliferation, migration and invasion of T24 cells transfected with pshRNA-survivin2 expression plasmid are inhibited. RNAi targeting survivin has a potential value in gene therapy of bladder cancer.