抑制具有PDZ结合域的转录共刺激因子表达对结直肠癌细胞株HCT116凋亡的影响及机制

Effect of transcriptional co - activator with PDZ - binding motif gene silencing by RNA interference on proliferation and apoptosis of colon cancer cell line HCT116 and mechanism

目的探讨RNA干扰技术沉默具有PDZ结合域的转录共刺激因子（TAZ）基因表达对结直肠癌细胞凋亡的影响及其机制。方法合成TAZ的小干扰RNA（TAZ—siRNA）以80nmol／L转染结肠癌细胞株HCT116，抑制TAZ表达；转染48h后细胞计数法（CCK-8）检测细胞的活性，流式细胞仪实验检测细胞凋亡率，实时定量反转录聚合酶链反应（RT—qPCR）和Westernblot检测转染各组HCT116细胞TAZ、B细胞淋巴瘤／白血病-2（bcl-2）、bcl-2相关X蛋白（bax）和细胞色素C（CytC）基闲mRNA和蛋白表达。结果TAZ—siRNA能有效沉默HCTI16细胞TAZ基因的表达（24h抑制率为40．5％，48h抑制率为82．5％，72h抑制率为69．8％）。有效抑制HCT116细胞中TAZ表达后，转染组细胞的活性（1．01±0．12）明显低于阴性对照组和COH—siRNA组（1．73±0．12和1．68±0．09）；凋亡率在TAZ—siRNA组、阴性对照组、con—siRNA组分别为（33．20±0．35）％、（11．674-0．12）％和（12．03±0．11）％。TAZ—siRNA转染后HCT116中TAZ和bcl-2基因和蛋白表达下调，而bax和CytC基因表达升高和蛋白的表达上调（P〈0．05）。结论利用RNA十扰技术沉默结直肠癌HCTI16细胞TAZ基因后可以显著抑制细胞的活性，并能明显促进细胞凋亡，其机制可能是TAZ通过调控线粒体相关凋亡途径影响细胞凋亡。

Objective To explore the proliferation and apoptosis of colon cancer cell line HCT116 cells by applying RNA interference technique to silence transcriptional co - activator with PDZ - binding motif （TAZ） gene. Methods Small interference RNA （siRNA） targeting TAZ was synthesized and trans- fected into HCT116 cells with concentration of 80 nmol/L. The transcription and translation levels of TAZ expression were detected by real -time quantitative reverse transeriptase -polymerase chain reaction （RT- qPCR）. The cell activity, proliferation and apoptosis of transfected tumor cells were determined by cell counting kit - 8 （ CCK - 8 ） and flow cytometry at 48 h after transfection. The gene and protein expres- sion levels of TAZ, B cell lymphoma/leukemia - 2 （ bcl - 2）, bcl - 2 associated X protein （bax） and cy- tochrome C （ Cyt C） were detected by RT - qPCR and Western blotting. Results TAZ expression was knocked down after siRNA trausfection （24 h： 40. 5% , 48 h： 82. 5% , 72 h： 69. 8% ）. After TAZ was suppressed by siRNA, the cell activity in TAZ - siRNA group （ 1.01 ±0. 12） was weaker than that in con- trol group and con- siRNA group （1.73 ± 0. 12, and 1.68 ± 0. 09 respectively）. The apoptosis rate in TAZ - siRNA group, control group, and con - siRNA group was （ 33.20 ± 0. 35 ） %, （ 11.67 ± 0. 12 ） %, and （12.03 ±0. 11 ）% respectively. The gene and protein expression levels of TAZ and bcl -2 were de- creased when TAZ was silenced, and the expression of bax and Cyt C increased. Conclusion TAZ gene silenced by siRNA can inhibit the cell activity and induce the apoptosis of colon cancer cell HCT116, which may be regulated though mitochondrial - associated pathway.