摘要
目的 通过RNA干扰技术下调VTCN1基因在人膀胱癌HT-1376细胞株中的表达,观察VTCN1基因表达下调后对膀胱癌细胞生物学行为的影响.方法 将针对VTCN1基因的小干扰RNA（siRNA）序列重组入真核表达载体中构建pU-VTCN1-siRNA,转染至膀胱癌HT-1376细胞株中,采用逆转录-聚合酶链反应（RT-PCR）、Western blot法检测转染pU-VTCN1-siRNA后的HT-1376细胞中VTCN1在基因和蛋白表达.采用流式细胞仪分析转染pU-VTCN1-siRNA后的HT-1376细胞的细胞周期,应用Transwell侵袭小室模型观察转染pU-VTCN1-siRNA后HT-1376细胞侵袭力.结果 转染pU-VTCN1-siRNA后的HT-1376细胞中,VTCN1的基因和蛋白表达水平显著下降（P＜0.05）,细胞周期被阻滞在G1期,G1期细胞从37.3％上升到74.6％,而S期细胞由46.36％下降到8.9％（P＜0.05）,Transwell侵袭实验显示侵袭细胞数由空载组（278 ±42）个和空白组（293±460个下降为实验组的（39±6）个,体外侵袭能力明显下降（P＜0.05）.结论 通过siRNA技术下调VTCN1基因表达能抑制HT-1376细胞增殖和侵袭能力.
Objective To study the effect of inhibitor of apoptosis VTCN1 down-regulation by small interfering RNA-mediated RNA interference （RNAi） on the biological features of bladder carcinoma cell line HT-1376.Methods HT-1376 cells were transfected with synthetic small interfering RNA （siRNA） targeting VTCN1.Expression of VTCN1 mRNA and protein were respectively measured by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting,transfection efficiencies were monitored by those ways.The distribution of cell cycle phases was determined using flow cytometry.The proliferative and invasive ability of HT-1376 cells in vitro was evaluated by the colony-forming unit assay and Transwell migration assay respectively.Results Both VTCN1 mRNA and protein expression were significantly decreased in the experimental group compared with controls （P ＜ 0.05）.The cell cycle was arrested in the G1 phase,and G1 phase cells increased from 37.3％ to 74.6％,while the S phase cells from 46.36％ down to 8.9％ （P ＜ 0.05）,Transwell invasion assay showed the number of invasive cells by the no-load group 278 ± 42 and 293 ± 46 control group dropped to experimental group,39 ± 6 （P ＜ 0.01）.Conclusion Silencing of VTCN1 gene inhibits cell proliferation and decreases invasion ability of HT-1376 cell.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第10期2126-2128,共3页
Chinese Journal of Experimental Surgery
