CLONING OF MAGE-n GENE AND ANALYSIS OF IT'S EXPRESSION IN HCC SAMPLES

It has been shown that MAGE-1 was preferentially expressed in hepatocellular carcinoma (HCC), which suggest that it could be a target in HCC immunotherapy. But it is still known a little about the message of the other MAGE family genes expressed in HCC. To understand the expression of the other MAGE genes would give help to more HCC patients to be accessible to this kind of therapy.In this research, MAGE-1 whole cDNA sequence, part of MAGE-6 and a new MAGE gene, which is named MAGE-n, were cloned by using molecular biology technique for further use in HCC immunotherapy. MAGE-n was then proved to be existent by RT-PCR and subsequent DNA sequencing. The expression of MAGE-n in HCC and non-cancerous tissues were analyzed by using in-situ hybridization technique. The HLA-A2 ^ A24 binding epitope was predicted by computer soft ware, which formed a base for later research of the use of MAGE-n in HCC immunotherapy. At the same time, the research provided data for improving the method of detecting the MAGE family gene. 1. The cloning of MAGE-n gene using RT-PCR methodMAGE-1 was cloned by RT-PCR method. The RT-PCR product contains 3 kinds of MAGE genes proved by DNA sequencing technique, which were:MAGE-1, part of MAGE-6 and a gene that was not same as any known genes, but highly homologous to MAGE-A family genes. It was named MAGE-n (novel and need to be known). A pair of primer was designed to clone part of MAGE-n in HHCC by RT-PCR method to verify the existence of MAGE-n gene. The PCR product was verified to be MAGE-n sequence, and MAGE-n was proved to be existence. Subsequently, RT-PCR was performed in the other 4 hepatocellular carcinoma cell lines, in which it was found to be expressed in HHCC, 7402 cell lines. It also proved the existence of the MAGE-n gene.2. Detection of the expression of MAGE-n gene in HCC tissues using in situ hybridizationTwo oligo-nucleotide probe was designed and synthesized according to the result of BLAST analysis to detect the expression of the MAGE-n gene in human HCC tissues sample, normal tissue and some different tissues on a human tissue array plate. It was detected in 10%(5/50) HCC samples. And no expression was detected in normal, liver cirrhosis and non-cancerous tissues, which suggest that MAGE-n could be potentially used as a target for immunotherapy for HCC.3. Prediction of HLA-A2, A24 binding epitope of MAGE-n gene productHLA-A2, A24 binding epitope of MAGE-n was predicted in MAGE-n protein by using the software based on web. It was shown that three epitopes of MAGE-n has been reported previously. There were some other highly scored predicted epitopes in the protein which suggest the potential of the use of these MAGE-n epitopes in HCC immunotherapy.