Abstract
Objective To observe the effects of over-expression of target gene MAGE-A3 by transient transfection on proliferation and metastasis of human hepatocellular carcinoma (HCC) cell lines.Methods MAGE-A3 target gene that containing restriction enzyme cutting site of NheI and KpnI was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from HCC tissues.The target gene was cloned into the expression vector pcDNA3.1 ( - ) to construct MAGE-A3 eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3,which was checked by restrictive enzyme digestion and DNA sequencing,and then transiently transfected into human HCC cell line SMMC7721 with Lipofectamine 2000.The mRNA and protein expression of MAGE-A3 was detected by semi-quantitive RT-PCR and Western blotting assay respectively.MTT assay and Boyden chamber assay were performed to detect the effects of MAGE-A3 on colony formation and metastasis.Results MAGE-A3 target gene was amplified and the eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3 was correctly constructed,and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western blotting revealed a strongly increased level of MAGE-A3 mRNA and protein expression in transfection group as compared with untransfected group and empty vector-transfected group (for MAGE-A3 mRNA expression level: 7838,2847 and 1557 in three groups,and for MAGE-A3 protein levels: 30 938,24 088 and 29 654,respectively).Both the number of HCC cells transfected by peDNA3.1-MAGE-A3 in MTT assay and the number of cells penetrating matrigel were increased as compared with untransfected group and empty vector-transfected group (P ＜0.05 ).Conclusion The eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3 was successfully constructed.The number of cells and invasiveness of the HCC cells transfected by pcDNA3.1-MAGE-A3 were significantly increased,suggesting MAGE-A3 gene may play a significant role in proliferation and metastasis of human HCC. Key words: MAGE; Transfect; Carcinoma,hepatocellular; Proliferation
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