| In our previous research, we used multi-tissues Northern blot to prove that the expression tendency of BC047440 gene significantly increased in tumor tissues. In addition, the expression of BC047440 gene in human hepatoma is higher than that of corresponding normal tissues. Thus BC047440 gene is associated gene in the carcinogenesis and progression of hepatocellular carcinoma. therefore, we inhibit the expression of BC047440 gene with RNAi to research growth characteristics of HepG2 of human hepatocarcinoma cells.RNA interference is a kind of conservative response of defense living nature existed. It is a complex process involving many factors and many steps. This process maybe is divided into these several stages. Double-stranded RNA was cut about 21~23nt siRNA by endonuclease RNaseⅢin cell, and formatted RNA-induced silencing complex, RISC identified and degraded mRNA of homological rank, it leads to ultimately specific silence of gene expression. To construct carrier that it can transcript functional siRNA in cell, and it was advantaged to confuse expression of BC047440 gene to establish background for further investigation of gene function and therapeutic tool.Objective: RNA interference (RNAi) has recently been used to silence gene expression in various species, it can inhibit evidently expression of objective gene. The connection between siRNA of BC047440 gene and plasmid pGenesil-1 of fluorescent protein expression, the constructing siRNA recombinant plasmid and establishing the cell lines of stable inhibition of BC047440 gene by siRNA in hepatocarcinoma cells.on this basis, the roles of BC07440-siRNA on the cell proliferation ability, cell-cycle and apoptosis were studied, which can provide a new target and experiment data for gene therapy on HCC.Method:(1) Designing the BC047440 specific siRNA on web with bioinformatics technology and constructing the pGenesil-1-BC047440-siRNA in vitro. (2)After separately transfection with pGenesil-1 and pGenesil-1-BC047440-siRNA, and selection with G418, two stable transfection cell lines were builted, which were renamed with PP1 and PP2 separately, the control group was hepatocarcinoma HepG2 cell without any disposal were renamed with PP0. (3) Through realtime fluorescent quantitative PCR detects mRNA level expression of BC047440 gene after RNAi .(4) the cell proliferation and cloning efficiency was measured by CCK-8, the plate colony formation assay separately . Using Hochest 33342 for staining the nucleus and then observes the nuclear morphological change to dectet cell apoptosis. Finally cell cycle was performed by FCM .Results:The recombinant vector was demonstrated to be right through digestion with the corresponding enzymes and agarose gel eletrophoresis; we obtained stable transfectants; endogenous BC047440 gene expression could be efficiently down-regulated by transfection of siRNA BC047440 gene in cultured HepG2 cell, the cell growth rate in PP2 groups were significantly inhibited compared to those in PP0 and PP1 groups detected by CCK-8. Meanwhile the cell colony formation rates were statistically decreased in siRNA treated groups. Which were [(39.31±5.39)%] in PP1 and [(40.96±3.21)%] in parental group PP0 , [(8.420±0.82)%] in PP2, (p<0.01,vs PP1,PP0 groups), after using Hoechst 33342 for staining the nucleus separately we never observed that morphological change in all groups showed the characteristic of apoptosis. Using FCM, it shows that S-phase cells PP2 group compared to those in PP0 and PP1 (p<0.05), contrasted between normal control groups(p>0.05) reveal that cell percentage of S-phase in PP2 was apparently more than that in the PP1,PP0 groups. Cell percentage of G1-phase in PP0 was slightly higher than that in PP1,PP2 (p<0.05),contrasted between PP1,PP2(p>0.05)。Conclusion:the shRNA designed and synthesized were connected into expression vector correctly; endogenous BC047440 gene expression could be efficiently down-regulated by transfection of siRNA BC047440 gene in cultured HepG2 cell after RNAi, the HepG2 cell growth rate were significantly inhibited by interfering experession of BC047440 gene. The mechanism of proliferation inhibition is not through the way of promoting the apoptosis but rationalized by the cell cycle which is arrested in S phase.The experiment of biological activity certificates that BC047440 gene expression was evidently decreased and growth of cell was evidently inhibited after RNAi. BC047440- siRNA may be used as a novel method to inhibit the proliferation of hepatoma carcinoma cell.
|
PDF Full Text Request