siRNA对子宫颈癌SiHa细胞VEGF-165表达影响的研究

Inhibitive effect of siRNA on VEGF-165 expression in human cervical cancer SiHa cell lines

目的:探讨小分子干扰RNA(siRNA)对子宫颈癌细胞系SiHa细胞中VEGF-165的表达及细胞增殖的影响。方法:体外化学合成针对VEGF-165的siRNA,在转染试剂的介导下转染SiHa细胞。实验分为3组,设未转染细胞为空白对照组、VEGF-165特异性si RNA转染的实验组及无关序列siRNA转染的阴性对照组。利用MTT法检测SiHa细胞转染后细胞增殖活性,荧光实时定量RT-PCR法分别检测转染12、24、48和72h后SiHa细胞中VEGF-165 mRNA的表达情况,ELISA法检测细胞培养液中VEGF-165蛋白分泌量的变化。结果:VEGF-165 siRNA转染SiHa细胞后,各时间点与对照组相比VEGF mRNA表达水平明显下降(F值分别为99.94、169.27、460.90和115.86,P<0.05);VEGF蛋白分泌量明显减少,差异有统计学意义(F值分别为107.06、136.46、147.10和54.25,P<0.05);细胞生长较对照组明显受到抑制(F值分别为70.58、61.17、113.39和100.94,P<0.05)。而阴性对照组与空白对照组比较,上述指标差异均无统计学意义,P>0.05。结论:VEGF si RNA可以有效抑制子宫颈癌Si Ha细胞VEGF mRNA及其蛋白的表达水平,抑制细胞增殖,为子宫颈癌的基因治疗提供依据。

OBJECTIVE： To study the effect of siRNA on VEGF-165 expression in vitro on cultured SiHa cells. METHODS： The specific siRNA was transfected into VEGF overexpressed cervical neoplasms cell SiHa lines in vitro by Hiperfect transfection reagent. The VEGF-mRNA level was detected by real time RT-PCR after the transfection with siRNA. The effect of RNA interference was observed at 12, 24, 48 and 72 hours, The secretion of VEGF protein in the supernatant was evaluated by ELISA. The cell proliferation was tested by MTT assay. RESULTS： Sequence specific siRNAs of VEGF-165 effectively downregulated the mRNA levels of VEGF compared with the controis （F were 99.94, 169.27,460.90 and 115.86, P〈0.05）, The protein levels were down regulated obviously post transfection （F were 107,06, 136.46, 147.10 and 54.25, P〈0.05）. The growth of SiHa cells was obviously inhibited （F were 70.58,61. 17,113.39 and 100, 94, P〈 0.05）. The cells in control groups had not obviously changed, P〉0.05. CONCLUSIONS： Specific siRNA can effectively downregulate the expressions of VEGF mRNA and its protein, and inhibit the cell growth. The study makes the therapeutic approach with gene therapy more possible in cervical neoplasms.