The Proliferation Effects Of SiRNA Silencing Of The CIP2A Gene On Human HepG2Cell

ObjectiveCancerous inhibitor of protein phosphatsase (CIP2A) is an inhibitor of proteinphosphatase2A in cancer. To knockdown the CIP2A gene expression in human hepatoma cell line HepG2by using RNA interference technology, The five CIP2A gene interference fragments and a fragments without CIP2Agene interference have been designed, and an interference fragment with the best silencing efficiency was obtained. Firstly. siRNA was stably transfected into HepG2cells using puromycin to obtain stable expression cell lines. In order to explore the biological effects of CIP2A gene on human HepG2cells, Western blot, RT-PCR and MTT approaches have been used to investigate the relationship between c-Myc and CIP2A.Methods1The plasmid with interference fragment was transformed into competent cells of E.coli. The plasmid DNA was digested by double restriction enzymes, and sequenced to determine the correctness of interference fragment.2The plasmid with interference fragment was transfected into human hepatoma cell line HepG2using Lipofectamine2000. The best siRNA sequence which can silent CIP2A was selected from five siRNA sequences which were specifically targeting CIP2A.3The best selection of puromycin concentration:The optimal concentration of puromycin for screening stable cell lines was determined.4Stably expressing cell lines:The best interference plasmid of silencing CIP2A gene was stably transfected into human hepatoma HepG2cells by using puromycin for obtaining stable cell lines.5The effect of CIP2A gene silence on c-Myc gene:After the CIP2Agene being silenced, c-Myc protein expression and the c-Myc mRNA expression were respectively detected by Western blot and RT-PCR.6The biological effects on human hepatoma HepG2cells after CIP2A gene silencing:The growth of HepG2cells after CIP2A gene be silenced was detected by MTT method.ResultsCIP2A plasmid DNA was transformed into E.coli, and further was extracted. The plasmid DNA with interference fragments were confirmed by restriction enzyme digestion and DNA sequencing. Finally, the best sequence (plko.1-2) was determined by using transiently transfected detection of transiently transfected cells of CIP2A the protein and mRNA expression, plko.1-2was transfected into HepG2cells with puromycin to obtain stable cell lines. RT-PCR results showed that the CIP2A gene mRNA expression in plko.1-2group was significantly different compared with the blank control group (P<0.01); in plko.1-2group c-Myc gene mRNA expression was not statistically significant compared with the blank control group and plko.1-control group (P>0.05); Western blot analysis results show that when the plko.1-2group was compared with the blank control group and plko.1-control group the CIP2A protein expression was significantly lower (P<0.01), and when the plko.1-2group was compared with the blank control group and plko.1-control group the c-Myc protein expression was significantly lower (P<0.01):MTT assay results showed that the cell’s ability to reproducing was decreased after siRNA silencing of CIP2A gene.ConclusionThe plasmid DNA with interference fragment was transformed into competent cells of E.coli. Plasmid DNA was extracted to determine the correceness of the interference fragment by double restriction enzyme digestion and sequencing. After transient transfection, protein and total RNA were extracted. Western blot and RT-PCR results have showed that CIP2A gene may impact on c-Myc gene expression in post-transcriptional level. MTT assay results have also indicated thatCIP2A gene suppression can inhibit tumor cell growth.