Effects of adenovirus-mediated PSMA3 gene overexpression on proliferation，cell cycle and apoptosis of human hepatocellular carcinoma SMMC7721 cells

Abstract:

Objective：To construct the adenovirus vector of proteasome subunit alpha type 3（PSMA3） gene and investigate whether the adenovirus vector carrying PSMA3 gene affecting the proliferation，cell cycle，apoptosis and the growth of nude mice bearing tumors of human hepatocellular carcinoma cell line SMMC7721. Methods：cDNA fragment encoding human PSMA3 gene was amplified by re－verse transcriptase-polymerase chain reaction（RT-PCR）. The PCR amplified fragment was first cloned into pTG19-T vector，and then subcloned PSMA3 gene into the shuttle vector pAd－Track-CMV for constructing the recombinant plasmid pAd－Track/PSMA3，which homologously recombinated with the ade－noviral backbone vectors Adeasy-1 to generate recombinant adenoviral plasmids Ad/PSMA3. The recombinant adenoviruses Ad/PSMA3 were generated by transfecting the recombinant adenoviral DNA into 293 cells and then employed to infect SMMC7721 cells. The expression of enhanced green fluorescent protein（EGFP） in transfected SMMC7721 cells were observed by fluorescence microscope. The proliferation of transfected SMMC7721 cells were tested via Cell count kit-8（CCK-8），and the expreesion levels of PSMA3，proliferating cell nuclear antigen（PCNA） and caspase-3 mRNAs proteins in transfected SMMC7721 cells were detected by RT-PCR and Western blot，respectively. The cell cycle distribution and apoptosis rate of transfected SMMC7721 cells were analyzed by flow cytometry（FCM）. SMMC7721 cells infected with pAd/PSMA3 virus were transplanted subcutaneously into nude mice to gen－erate tumors，and the growth of the tumors was observed day by day. Results：PSMA3 gene was successfully cloned. Adenoviral virus particles which possess infectious competent were produced from Ad/PSMA3. The results of CCK-8 revealed that the proliferation of SMMC7721 cells，which overexpressed PSMA3，were slower than the control cells，especially from 48 h after transfection（P<0.01）. The results of RT-PCR and Western blot demonstrated that SMMC7721 cells transfected with pAd/PSMA3 recombinant virus can effectively overexpressed PSMA3，and the expression of PSMA3，caspase-3 mRNA and protein were up-regulated，while the expression of PCNA mRNA and protein were down-regulated in the cells. FCM detection confirmed that SMMC7721 cells transfected with pAd/PSMA3 were blocked in the G1 phase of cell cycle，and promoted apoptosis，and the difference of the apoptosis rate between trans－fected and control cells were statistically significant（P<0.01）. Conclusion：PSMA3 gene was successfully cloned. The proliferation of SMMC7721 cells，which overexpressed PSMA3，can be effectively suppressed，and their apoptosis be promoted. The growth of nude mice bearing tumors of SMMC7721 cells overexpressed PSMA3 can be also inhibited. These effects may be related to the down-regu－lattion of the PCNA expression and the up-regulation of the caspase-3 expression in the cells.