Influence Of Silencing SnoN Gene By SiRNA On Proliferation And Apoptosis Of HepG2Cells

[Objective]The SnoN gene was discovered as an oncogene in1980s. It is a nuclear protein belonging to the Ski family. SnoN showed many biological activities founded by many researchers home and abroad. SnoN can effect embryonic development, adult tissue differentiation and cancer progression significantly. The researches on the role of SnoN in tumors in recent years become hot. These researches mainly concentrated in esophageal cancer, pancreatic cancer, leukemia, melanoma, lung cancer, breast cancer, colon cancer, but the role in liver hepatocellular carcinoma is still not clear. The siRNA （small interfering RNA） technology is used in our study to down-regulation SnoN gene expression of HepG2cells, then detect the changes of proliferation, apoptosis and other biological characteristics. This study can provide a theoretical basis and therapeutic targets for pathogenesis research and gene therapy of hepatocellular carcinoma.[Methods]（1） Human hepatoma HepG2cells was recovered and cultured to the logarithmic growth phase.（2） Reference SnoN sequence （NM₀05414） in GeneBank. Design and synthetize three disruption sequence targeting SnoN gene, one nonspecific negative control sequences, and one nonspecific sequences labeled by FAM fluorescent according to the design principle of siRNA by the Ji Ma Corporation of Shanghai. （3） HepG2cells was cultured. FAM-siRNAs with different concentration were transiently transfected into HepG2cells by cationic liposome Lipofectamine2000TM. FAM fluorescence expression of each group were detected by Flow Cytometry to select the best transfer concentration.（4） The mRNA expression of SnoN was detected by reverse transcription-polymerase chain reaction （RT-PCR） while the protein expression was detected by western blot, then compared interference efficiency of the three siRNAs. The siRNA with the highest interference efficiency was selected.（5） Detected the HepG2cell proliferation of blank group, negative control group and the interference group by using Cell Counting Kit-8.（6） Detected the HepG2cell apoptosis of blank group, negative control group and the interfering group by using flow cytometry （The Alexa Fluor（?）488annexin V/Dead Cell Apoptosis Kit）.[Results]（1） The siRNA transfection efficiency test showed that the optimal transfection efficiency is84.8%, and the optimal transfection concentration is100nmol/L. Use the same conditions for subsequent transfection experiments.（2） The results of RT-PCR and Western blot showed that the mRNA and protein expression levels of SnoN in the three groups were decreased significantly compared to the blank group and negative control group. The best interference effect was found in siRNA-C group, and the inhibiting efficiency can reach to62.4%. No significant difference can be found between the blank group and negative control group.（3） The proliferation rate of interference group slowed down significantly with CCK-8compared to blank group and negative control group. The most inhibiting effects appeared at the third day after transfectd.（0.687vs1.548,1.378, P<0.05）.（4） The results of flow cytometry showed that the early apoptosis of interference group is increased significantly compared to blank control group and negative control group after transfected48hours.（9.84%vs0.65%,1.78%, P<0.05）.[Conclusions]SiRNA can be transfected effectively by cationic liposomes. The transfecting efficiency can up to84.8%while siRNA concentration is100nmol/L. The three siRNAs targeting the SnoN gene have certain interfering effect, and they can inhibit the mRNA and protein expressing level of SnoN effectively. The proliferation of HepG2cells was slow down and cell apoptosis was increased after down-regulating of SnoN gene in HepG2cells. Our study shows that SnoN gene plays an important role on the occurrence and development in hepatocellular carcinoma. SnoN may be a new target for the treatment of hepatocellular carcinoma.