A Study On Anti-hepatocellular Carcinoma Activities By Survivin Targeted SiRNA Nano Liposome

Objectives This study aims to investigate the activities of survivin targeted small interfering RNA(siRNA) on gene expression, the proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H as well as anti-hepatocellular carcinoma in vivo in mice.Methods Survivin specific siRNA was designed and synthesized. Then siRNA/ liposome complex was transfected into hepatocellular carcinoma cell line MHCC-97H. The MHCC-97H cells were divided into group of survivin siRNA(Sur-siRNA), negative control siRNA group(NC) and blank group(Normal). Survivin mRNA and protein expression were detected by RT-PCR and Western blot respectively. The proliferation of MHCC-97H was measured by MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay. The Annexin V/PI double labeled flow cytometry was employed to measure the apoptosis at 24h after transfection in different concentrations of survivin siRNA(12.5,25.0,50.0 respectively). On the other hand, after hepatocarcinoma nude mice model was constructed, PEG-Survivin siRNA was injected intravenously into mice. Compared with control group, the tumor volume, survivin mRNA level and protein expression were detectedResults In vitro experiment, after 48 hours of transfection, the mRNA levels were 0.55±0.16(Si-survivin),0.85±0.28(NC) and 0.93±0.40(Normal), and there was significantly different statistically in three groups(F=414, P<0.01). The level of Survivin mRNA was the lowest in Si-survivin group, which was statistically different with the other two groups(t=-20.56,-28.37, P<0.001). The levels of Survivin protein expression in three groups were 0.602±0.005 (Si-survivin),0.835±0.007(NC) and 0.993±0.003(Normal) at 48h after transfection, and there was different statistically in three groups(F=238, P<0.01). The lowest level of protein expression was in Si-survivin group, which was statistically different with the other two groups(t=-40.17,-66.03, P<0.001). After 72 and 96 hours of transfection, the inhibitory rate of cell growth was higer significantly in Si-survivin group[(19.5±3.6)%,(12.0±0.9)%] compared with NC group[(3.6±0.9)%,(-1.3±6.1)%](t=36.18,42.53, P<0.05). The apoptosis rates in 12.5nmol/L,25.0nmol/L,50.0 nmol/L survivin siRNA were (22.64±2.54)%, (35.37±3.28)%, (53.28±4.35)%, respectively. However, in NC and Normal group, the apoptosis rates were (8.77±1.25)% and (9.72±1.37)%. The rates were statistically different in those five groups(F=35.93, P<0.01). And in the three concentrations of siRNA groups, the rates were statistically different (t=-29.73、-38.57, P<0.001).In mice model, tumor volume was significantly smaller in group of survivin siRNA injection(SU-Ⅳ group) after 10 days of injection(P<0.05). Survivin mRNA and protein expression level were lower in SU-Ⅳ group (P<0.001)Conclusion In summary, siRNA specific to survivin is capable of inhibiting the proliferation and therefore inducing the apoptosis of MHCC-97H cells by down regulating survivin gene expression. SiRNA targeting survivin can down-regulate survivin mRNA and protein expression in mice model of hepatocellular carcinoma and inhibit the growth of tumor in vivo.