肝細胞癌細胞株MHCC97Hのサバイビン発現,増殖およびアポトーシスに及ぼす低分子干渉RNAの効果【Powered by NICT】

Abstract

Objective To observe the effect of small interfering RNA(siRNA)targeting Survivin gene on urviving expression, proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H. Methods Survivin sequence specific siRNA was designed and synthesized.siRNA/liposome complex was transfected into hepatocellular carcinoma cell line MHCC- 97H.The MHCC-97H cells were divided into Survivin siRNA group(Si-survivin), negative control siRNA group(NC group)and blank group(normal control group). Survivin mRNA and protein expressions were detected by reverse transcription-PCR and Western blot, respectively.The proliferation of MHCC-97H was measured by methythiazolydiphenyl-tetrazolium bromide assay.The Annexin V/PI double labeled flow cytometry was employed to measure the apoptosis at 24 h after transfection in different concentrations of Survivin siRNA(12.5, 25.0 and 50.0 nmol/L, respectively). Results After 48 h of transfection, the Survivin mRNA levels were 0.55±0.16(Si-survivin group), 0.85±0.28(NC group)and 0.93±0.40(normal control group), respectively, which were significantly different among 3 groups(F=414, P<0.01). The level of Survivin mRNA was the lowest in Si-survivin group, which was statistically different with NC group and normal control group(t=-20.56, -28.37, all P<0.001). The levels of Survivin protein expression in 3 groups were 0.602±0.005 (Si-survivin group), 0.835±0.007(NC group)and 0.993±0.003(normal control group)at 48 h after transfection, which were statistically different among 3 groups(F=238, P<0.01). The lowest level of protein expression was in Si-survivin group, which was statistically different with NC group and normal control group(t=-40.17, -66.03, all P<0.001). After 72 h and 96 h of transfection, the inhibitory rate of cell growth was significantly higher in Si-survivin group[(19.5±3.6)%, (12.0±0.9)%] compared with that in NC group[(3.6±0.9)%, (-1.3±6.1)%](t=36.18, 42.53, all P<0.05). The apoptosis rates in 12.5, 25.0, 50.0 nmol/L Survivin siRNA were (22.64±2.54)%, (35.37±3.28)% and (53.28±4.35)%, respectively.However, in NC group and normal control group, the apoptosis rates were (8.77±1.25)% and (9.72±1.37)%.The rates were statistically different among those 5 groups(F=35.93, P<0.01). And in the apoptosis rates of siRNA groups in different concentrations were statistically different when compared between each two groups(t=-29.73, -38.57, all P<0.001). Conclusion Survivin specific siRNA can inhibit the proliferation and induce the apoptosis by blocking Survivin gene expression in hepatocellular carcinoma cell line MHCC-97H. Key words: Hepatocellular carcinoma; Survivin; Small interfering RNA