Study On The Mechanism Of HepG2 Proliferation Regulated By BC047440 Gene

The hepatocellular carcinoma is one of most common malignant tumors at our Country, and the present treatment stategy is not ideal. Although great effort has been made, the pathogenesis of hepatocellular is still largely unknown BC047440 highly expressed in hepatocellular carcinoma , is a novel gene previously identified by us through SSH. We found that the expression of BC047440 in hepatocellular carcinoma was higher than that in their adjacention cancerous tissue,and this gene is also relevant to the proliferation of hepatocellular carcinoma. As NF-κB(nuclear factor-kappaB) is recently reported to be essential for the development of hepatocelluar carcinoma, we would like to investigate whether BC047440 gene regulates HepG2 proliferation through NF-κB. In addition, we also would like to investigate how the BC047440 gene regulates NF-κB activation through comparation of HepG2 cells and HepG2 cells BC047440 gene silenced using 35K Human Genome Array.Objective: To investigate whether BC047440 gene could regulate HepG2 proliferation through enhancing NF-κB activity and to analyze the differential expression gene between HepG2 cells and HepG2 cells BC047440 gene silenced by RNAi using 35K Human Genome Array, to investigate the mechanism that BC047440 gene regulate HepG2 proliferation and understand the effection of BC047440 in hepatocellular carcinoma.Methods: (1) Three BC047440 gene-specific short hairpin RNA (shRNA1 , shRNA2 and shRNA3), together with negative shRNA HK, were designed and directly cloned into EGFP reporter plasmid pGenesil-1 and constructing the pGenesil-1-BC047440-siRNA in vitro. (2) Expression of BC047440 gene was investigated through quantitive RT-PCR analysis after BC047440 gene-specific shRNA1,shRNA2 and shRNA3 plasmids were successfully transfected into HepG2;(3) The proliferation of HepG2 cells were detected by MTT assay;(4) the protien of NF-κB signal pathway was determinen by Western Blotting; (5 ) Nuclear translocation and NF-κB activity was evaluated by electrophoretic mobility shift assay ( EMSA) and luciferase reporter assay;(6) The differential expression gene between HepG2 cells and HepG2 cells with BC047440 gene silenced was analyzed by 35K Human Genome Array, and the interesting candidate genes were confirmed by RT-PCR.Results: (1) Three expression vectors: Pgenesil-1-BC047440-1- siRNA(stated at 469nt),Pgenesil-1-BC047440-2-siRNA(stated at 572nt) and Pgenesil-1-BC047440-3- siRNA(stated at 616nt) was confirmed to be successfully constructed with the assay of enzyme digestion and sequencing.(2) Quantitive RT-PCR analysis showed Pgenesil-1-BC047440-1-siRNA,Pgenesil-1-BC047440-2 -siRNA and Pgenesil-1- BC047440-3-siRNA could specifically inhibit the expression of BC047440 gene in HepG2 by 80.22% ,58.63%,74.22%, respectively, in HepG2/BC047440-siRNA cell compared with that in HepG2 cell and in HepG2/HK cell (3) The Pgenesil-1-BC047440-1-siRNA, with highest silence effect, was found to be effectively inhibit the proliferation of HepG2 cells. in HepG2/BC047440-siRNA cell compared with that in HepG2 cell and in HepG2/HK cell on D1(0.011±0.002 VS 0.025±0.005 , p<0.01),D2(0.025±0.005 VS 0.044±0.006, p<0.01),D3(0.124±0.007 VS 0.168±0.013, p<0.01),D4(0.261±0.042 VS 0.406±0.077, p<0.05 ).(4) The level of nuclear translocation of NF-κB (5) Western Blotting analysis revealed decreased expression of p50 in silicening HepG2 compared with that of wild HepG2. (6) The ratio of luciferase/ Renilla Luciferase in silicening HepG2 is only 25% of wild HepG2. (7) Among the total 35000 probe sets, the expressions of 59 genes were down-regulated exceed 50% and 130 cDNAs up-regulated more than 2 fold in the silencing group when compared with normal controls.(8) Most of regulated genes belong to the groups that are important in maintaining cell division,cell cycle,cell apoptosis, signal pathway, histone,potassium/sodium channel, drug resistance,DNA repair et al.(9) TRAF6 mRNA was decreased in silicening HepG2 compared with that of wild HepG2 by RT-PCR, similar to human genome array.Conclusions: (1) Pgenesil-1-BC047440-siRNA expression vector was successfully constructed.It could effectively inhibit the expression of BC047440 gene,the inhibition ratio was about 80.22%,and it suggest that it could silence BC047440 gene.(2) The Pgenesil-1-BC047440-siRNA could effectively inhibited the proliferation of HepG2 cells, consistent with our earlier period findings.(3) BC047440 can regulate HepG2 proliferation through NF-κB signal pathway.(4)The results of human genome array suggest that BC047440 gene directly or indirectly regulate cell division,cell cycle,cell apoptosis,signal pathway,potassium/Sodium channel,drug resistance,DNA repair,and so on. The down-regulation of TRAF6 mRNA was verified by semi-quantitive RT-PCR ,and the result suggested that BC047440 gene might regulate NF-κB signal pathway inderectly by TRAF6.