Expression Of MAGE-A3Gene In Hepatocellular Carcinoma And Immuno-protective Effect To HCC

ObjectiveBy detecting the expressin of MAGE-A3gene in HCC, construting murinepcDNA3.1(+)-MAGE-A3eukaryotic expression vector and building mice hepatomamodel with murine hepatoma cell H22, we meat to provide the theoretical basis forimmune treatment and prognosis judgment in hepatocellular carcinoma(HCC)patients., and discusse MAGE-A3gene vaccine immune-protection effect to HCC.Method1. The expression of MAGE-A3gene in HCC and tumor-adjacent liver tissuespecimens from the First Affiliated Hospital of Fujian Medical University wasdetected using reverse transcriptase polymerase chain reaction (RT-PCR), tissue chiptechnology, immunohistochemistry. Then we analysised the relationship between thepositive rate and HCC patients clinical, biochemical, pathological dicators.2. Mice MAGE-A3gene (C terminal containing6x His tags) was synthesized bychemical means. Then the gene was cloned to pcDNA3.1(+) and amplified in E. coliDh5α. was obtained and detected using digestion identification and sequencingappraisal.3. The recombinant eukaryotic expression vector was transfected into293T cells, ahuman renal epithelial cell line, and the expression of MAGE-A3was detected byWestern-blot.4. Expressing of MAGE-A3gene in H22cell was induced by5-Aza-Cdr. Micewere immuned in muscle injection method3times,interval7days. The MAGE-A3gene positive expression H22cell was injected into mice axillary subcutaneous. Thetumoma growth wsa observed every day and the MAGE-A3antibody was detected byELISA assay.Results1. The expression rate of MAGE-A3mRNA in HCC was64.29%(36/56), obviously higher than in tumor-adjacent live tissues and nomal liver tissues whichwere0%(P <0.01). The expression rate of MAGE-A3antigen protein in HCC was53.9%(76/141), obviously higher than in tumor-adjacent live tissues and nomal livertissues which were0%(P <0.01).2. The recombinant eukaryotic expression vector pcDNA3.1(+)-MAGE-A3wasobtained and it was confirmed that MAGE-A3cDNA was inserted into the eukaryoticexpression vector correctly using digestion identification and sequencing.3. It was showed that recombinant eukaryotic expression vector pcDNA3.1(+)-MAGE-A3gene had been transfected into293T cells and the result was detectedby Western blot.4. The expression of MAGE-A3gene in H22cell cultured in the medium with5-Aza-Cdr can be detected by RT-PCR.5. The tumor nodus in null vectors and saline group mice were checked in9thdayafter innoculation and that in the recombinant vector group were checked in11thday.In15thday, mice were killed. The tumor nodules＇volume and weight in recombinantvector group was higher than in the other two group(P<0.05) The concentration ofMAGE-A3antibody was detected in mice blood serum by ELISA assay.Conclusion1. There was high expression rate of MAGE-A3genes in HCC. The expression ofMAGE-A3gene in HCC was not associated with tumor size, AFP, HBsag, recurrence,the background of liver cirrhosis, envelope and the degree of tumor differentiation.The expression of MAGE-A3gene in HCC was positively related to microscopictumor thrombus.2. The recombinant eukaryotic expression vector pcDNA3.1(+)-MAGE-A3wasobtained successfully, it can effectively express MAGE-A3protein. in vitro.3. The pcDNA3.1(+)-MAGE-A3gene vaccine can inhibit MAGE-A3genepositive expression tumor and it can inmunity protect mice against hepatocelluarcarcinoma.