特异性小干扰RNA靶向沉默RIP1基因表达对人大肠癌Lovo细胞生物学行为的影响

Effect of siencing RIP1 by specific siRNA on the biological behavior of human colon cancer cell line Lovo

目的探讨RNA干扰沉默RIP1基因表达对人大肠癌Lovo细胞生物学行为的影响。方法将人大肠癌Lovo细胞常规分为空白对照、阴性对照组和实验转染组。体外化学合成RIP1基因序列特异性小干扰RNA(siRNA),Hegene介导转染人大肠癌细胞株Lovo细胞;采用逆转录-聚合酶链反应(RT-PCR)方法检测特异性siRNA对RIP1基因在mRNA水平上沉默效果;转染后采用噻唑蓝(MrI'r)比色法检测siRNA对细胞增殖的影响;流式细胞周期法检测siRNA对细胞周期的影响;Transwell试验体外检测细胞迁移和侵袭能力。结果成功转染RIP1 siRNA的大肠癌Lovo细胞,RT-PCR显示RIP1 mRNA和蛋白表达在相应的癌细胞中明显下降;与阴性对照组比较,细胞增殖明显抑制(P<0.05);G0～G1期细胞[(54.68±1.54)%]明显多于阴性对照组[(48.48±1.96)%]和空白对照组[(43.92±1.68)%];RNA干扰明显抑制Lovo细胞迁移力和侵袭力(21±2.731vs.43±2.064)。结论 RIP1 siRNA可有效抑制大肠癌Lovo细胞RIP1基因的表达,从而抑制肿瘤细胞增殖,促进细胞凋亡,抑制细胞迁移和侵袭,RIP1siRNA能有效调控Lovo细胞恶性生物学行为。

Objective To study the effect of small interference RNA （siRNA）silencing RIP1 on the biological behavior of human colon cancer cell line Lovo. Methods Lovo cells were divided into three groups ： the bland control group, the negative control group, the RIPI-siRNA group. Chemically synthesized siRNA targeting RIP1 （RIPI-siRNA） was transfected into Lovo cells with high metastatic potential by Hygene. Reverse transcription- polymerase chain reaction（RT-PCR） was used to semi-quantify the RIP1 mRNA level. Then proliferation of Lovo cells was determined by methyl thiazol tetrazolium （MTY） assay. Flow cytometry was used for cell cycle analysis. The activities of motility and invasion of Lovo cells were assessed by transwell chamber inwasion assay in vitro, respectively. Results In the RIPI-siRNA group, the RIP1 mRNA level was down-regulaed remarkably（P 〈 0. 05）, the abilities of proliferation were inhibited, the number of cells in GO-G1 phrase increased in RIPI-siRNA group [ （54. 68 ± 1.54） % ] in comparison with negative control [ （48.48 ± 1.96） % ] and blank control groups [ （43.92 ± 1.68）% ] ,the motility and inwation of Lovo cells were inhibited significantly （21 ± 2. 731 vs. 43 ± 2. 064）. Conclusions Silencing RIP1 could regulate the malignant biological behavrors of colon cancer cell line Lovo effectively.