Inhibitory Effect of siRNA Expression Vector Inhibiting IGF2 Gene on the Proliferation of Hepatoma Cell Line Huh-7 / 中国药房

ABSTRACT

OBJECTIVE:To investigate the inhibitory effect of siRNA expression vector inhibiting human insulin-like growth factor 2(IGF2)gene on the proliferation of hepatoma cell line Huh-7. METHODS:siRNA expression vector pGL3-hAFP-hTERT-siRNA3(“siRNA3”)which inhibited IGF2 gene by dual promoter regulation of recombinant human alpha-foetoprotein(hAFP)and human telomerase reverse transcriptase(hTERT)was transfected into the Huh-7 cell and normal hepatocyte L-02,and then a nega-tive control group(vector pGL3-hAFP-hTERT)and a blank control group were set up. IGF2 mRNA expression was detected by re-al-time fluorescent quantitative polymerase chain reaction 48 h after transfection into the cells in all groups;the activity of the cells by the microplate reader 0,24,48 and 72 h thereafter;and the cell cycle and apoptosis by the flow cytometer 48 h thereafter,and the changes in the protein levels of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in the cell were detected by Western blot. RESULTS:Compared with the negative control group and blank control group,IGF2 mRNA expression in the Huh-7 cell transfected with siRNA3 was obviously weaker;at 48 and 72 h after transfection,the activity of Huh-7 cell signigicantly reduced, Huh-7 cells at G1 phase obviously increased and those at S phase markedly decreased;the occurrence of early,late and total apopto-sis in Huh-7 cells apparently increased,and the protein expression of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in cells significantly weakened,with statistically significance(P0.05). CONCLUSIONS:siRNA which inhibited IGF2 gene by dual promoter regulation of recombinant hAFP and hTERT can specially inhibit IGF2 gene expression and the prolifer-ation of Huh-7 cells,which may be involved with down-regulated protein expression of cell proliferation-associated gene PCNA, cell cycle control-associated genes Cyclin E2,Cyclin D2 and Cdc2 and apoptosis regulation-associated gene Bcl-2 as a result of down-regulated IGF2 mRNA expression and protein expres-sion.