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目的探讨转录水平基因沉默(TGS)技术和转录后水平基因沉默(PTGS)技术对肝素酶(HPA)基因干扰效果及其对肝癌SMCC-7721细胞侵袭能力的影响。方法从HPA基因启动子区和编码区分别设计并合成TGS和PTGS的小干扰RNA(siRNA),并转染肝癌细胞SMMC-7721;实时半定量PCR和Western blotting检测TGS和PTGS siRNA转染后48、72和96h HPA的表达,并设空白组作为对照;Transwell小室实验检测干扰后SMMC-7721细胞的侵袭能力。结果 TGS和PTGS两种技术在转染48h后,从mRNA和蛋白水平上均能成功干扰HPA表达;转染后72h,PTGS组HPA恢复表达,而TGS组仍保持沉默;转染后96h,两组HPA均恢复表达。Transwell小室实验表明,TGS和PTGS组HPA基因均能使SMCC-7721的穿膜细胞数减少,TGS组效果更明显。结论 TGS技术沉默肝癌SMMC-7721细胞中HPA基因的能力优于PTGS技术,并使肝癌细胞的侵袭能力显著降低。
Objective To investigate the effects of transcriptional level gene silencing (TGS) and posttranscriptional level gene silencing (PTGS) on heparinase (HPA) gene silencing and its effect on invasion of hepatocellular carcinoma cell line SMCC-7721. Methods Small interfering RNA (siRNA) of TGS and PTGS was designed and synthesized respectively from the promoter and coding region of HPA gene and transfected into SMMC-7721 cells. Real-time semi-quantitative PCR and Western blotting were used to detect the expression of TGS and PTGS siRNA , 72 and 96 h HPA expression, and set the blank group as a control; Transwell chamber test to detect invasion of SMMC-7721 cells. Results Both TGS and PTGS could interfere with the expression of HPA at mRNA and protein level 48h after transfection. At 72h after transfection, the expression of HPA was restored in PTGS group and remained silent in TGS group. After transfection for 96h, Group HPA were restored expression. Transwell chamber experiments showed that HPA gene in both TGS and PTGS groups decreased the number of transmembrane cells in SMCC-7721 group and the effect was more obvious in TGS group. Conclusion The TGS technique has a better ability of silencing HPA gene in hepatocellular carcinoma SMMC-7721 cells than that of PTGS technology and significantly reduces the invasion ability of hepatoma cells.