Effects of small interfering RNA on Survivin expression, proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H / 中华实用儿科临床杂志

ABSTRACT

Objective To observe the effect of small interfering RNA(siRNA) targeting Survivin gene on survivin 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 concentratiom 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.