摘要
目的探讨应用RNA干扰技术沉默信号转导和转录激活因子3(STAT3)基因对膀胱癌T24及5637细胞的影响。方法针对STAT3 mRNA序列设计合成3对编码小干扰RNA(siRNA)的DNA模板,构建pGenesil-1-shRNA-STAT3重组质粒,转染人膀胱癌T24及5637细胞。通过半定量RT-PCR、Western印迹检测STAT3基因不同水平的表达情况,采用四甲基偶氮唑盐(MTT)实验、流式细胞仪检测转染后细胞增殖及细胞周期的变化。结果成功构建pGenesil-1-shR-NA-STAT3重组质粒,并成功转染T24及5637膀胱癌细胞;半定量RT-PCR、Western印迹检测结果显示,重组质粒实验组细胞的STAT3基因表达在RNA及蛋白水平上显著低于对照组(P<0.05);MTT实验、流式细胞仪检测结果显示,重组质粒实验组细胞的增殖明显受到抑制并出现凋亡现象。结论pGenesil-1-shRNA-STAT3转染T24及5637膀胱癌细胞后,可有效抑制STAT3的表达,并抑制膀胱癌细胞的生长及增殖。
Objective To explore the effect of signal transducers and activators of transcription 3 （STAT3） silenced by RNA interference （RNAi） technique on bladder cancer cells T24 and 5637. Methods Targeted at STAT3 mRNA sequence, three pairs of DNA templates encoding small interference RNA （siRNA） were designed and synthesized. The recombinant plasmid pGenesil-1-shRNA- STAT3 was constructed and transfected into T24 and 5637 cells. Semi-quantitative RT-PCR and Western blot were applied to detecting STAT3 gene expression. Methyl thiazolyl tetrazolium （MTT） assay and flow cytometry were applied to detecting the changes of cell proliferation and cycle. Results pGenesil-1-shRNA-STAT3 was successfully constructed, and transfected into T24 and 5637 cells. Semi-quantitative RT-PCR and Western blot analysis demonstrated that pGenesil-l-shRNA-STAT3 could significantly inhibit the expression of STAT3 in T24 and 5637 cells; MTT and FCM results showed that it could suppress the growth of T24 and 5637 cells. Conclusion pGeneSil-1-shRNA- STAT3 transfection into T24 and 5637 cells can significantly inhibit STAT3 expression, and while sup- press the growth and proliferation of T24 and 5637 cells.
出处
《国际检验医学杂志》
CAS
2008年第3期196-199,223,共5页
International Journal of Laboratory Medicine
关键词
转录激活因子类
膀胱肿瘤
RNA干扰
研究
Activating transcription factors, Bladder neoplasms
RNA interference
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