TSP50in Human Colon Cancer: Expression, Methylation In Promoter

Background and objective:The potential role of cancer/testis antigens has paid gradually attention due toits not expressing in normal tissue except for testis and mutant expressing in cancer,which may be applied to tumor immunotherapy in the future.TSP50, as ahypomethylation gene, is isolated from testicular tissue and breast cancer bymethylation-sensitive representational difference analytical technique. Previousresearch shows TSP50highly expressed in normal testis, breast cancer, lung cancer,but didn’t express in other normal tissues, suggesting TSP50a cancer/testisantigens(CTAs). Present study suggests The expression of the cancer/testis antigen isregulated by methylation status of gene promoter. In our study, TSP50is identifiedby immunohistochemical staining technique in human colon cancer tissue, then isdetected by RT-PCR method.Bisulfite direct cloning and sequencing detect bothmethylation levels of CpG island in promoter region whether expressing TSP50ornot in order to investigate the relationship between expression of TSP50and itsmethylation in promoter region.Methods:we collect48cases of colon cancer paraffin-embedded specimens, using theimmunohistochemical staining technique to detect expression level of TSP50protein.We also gather and extract total RNA from5cases of fresh colon cancer specimens,then respectively test TSP50mRNA expression level and screen TSP50positiveand negative in colon cancer by RT-PCR. We randomly selected each of positive andnegative samples, extracting tissue genomic DNA, using bisulfite direct cloning andsequencing to detect both DNA methylation in the region of promoter. Results:In the48cases of colon cancer specimens, including39cases ofWell-differentiated colon adenocarcinoma,3cases of moderately differentiated colonadenocarcinoma,2cases of poorly differentiated colon adenocarcinoma,4cases ofmucinous carcinoma. Immunohistochemical results show that45cases of colonadenocarcinoma express TSP50protein, positive rate is94%with no significantdifferences between different types of tumors.RT-PCR results show3out of5cases of fresh specimens express TSP50,2cases express negatively. The positive expression rate of TSP50mRNA is60%.Bisulfite cloning sequencing method display methylation level of TSP50expressing positively in colon cancer is significantly lower than that of TSP50expressing negatively. The methylation levels of CpG sites in1,2,4,5,6,7,8,12ofTSP50positive and negative in colon cancer are0-16.7%,50%-83.3%respectively.Conclusions:1. TSP50express highly in colon cancer. TSP50can be used as a marker inclinical identification of colon cancer and as a new target for immunotherapy.2. The methylation level in promoter of TSP50expressed positively issignificantly lower than that of TSP50express negatively in colon cancer. Themethylation of TSP50in promoter regulate the expression of TSP50mRNA.