Study On Expression Of Cancer-testis Antigen TSP50and Mechanism In Gastric Cancer

Malignant tumor is still harmful to human health seriously，the main reasons islack of effective treatment。As the development of disciplines related to medicine，likeImmunology，Immune treatment becomes a new method to treat tumor。An idealtumor antigen for immunotherapy must be stable, the specific expression in tumorcells, does not exist in normal tissue，and it is essential to the genesis and developmentof tumor。Cancer-testis antigens possess this characteristic，therefore，they becomethe hot spot of research。Cancer-testis antigens are a group of tumor antigens，expressed in normal testisand different types of tumors because of their highly tissue restricted expression。Theyhad an important role in the genesis of tumor、proliferation of tumor cells、inhibitapoptosis、metastasis，and so on。Besides，The majority of cancer-testis antigen areimmunogenic，they had an important role in anti-tumor by inducing humoralresponses and cellular immune responses。Although，normal testis expressed cancer-testis antigen，because of existing of blood-testis barrier and the lack of humanleukocyte antigen (HLA) class I expression on the surface of germ cells，they couldnot present antigens effectively，so they do not cause autoimmune reaction。Discoveryof cancer-testis antigen has led directly to the development of antigen-specific cancervaccines，provide a new chance for Immune treatment of tumor。CTAs are consideredas promising target molecules for immunotherapy.It is accepted that regulation of DNA methylation has an important role in thecontrol of expression of CTAs。In eukaryotes，DNA methylation take place in CpGisland，CpG island mainly distribute in the area of promoter and the first exon。DNAmethylation has close relationship with genesis and development of tumor。Thetestes-specific protease50(TSP50) gene was a hypomethylated DNA fragment isolated from human breast cancer and human normal testes by the methylationsensitive-representational difference analysis technique。Previous researches showthat TSP50did not express in many normal tissues，but highly expressed in normaltestes、breast cancer and colorectal carcinoma。We speculate that TSP50is a cancer-testis antigen，and，the TSP50gene promoter’s DNA methylation pattern is associatedwith expression of TSP50。In this experiment，we detect the expression of TSP50ingastric cancer by Immunohistochemical staining and RT-PCR，and apply to themethylated sites sequencing to study the correlation between methylation status inpromoter and expression of TSP50。1、TSP50express in gastric cancer：In this experiment，we use Immunohistochemical staining and RT-PCR to testexpression of TSP50in gastric cancer from protein level and mRNA leverrespectively。We collect51cases of gastric cancer paraffin-embedded specimen，including adenocarcinoma of the stomach、squamous cell carcinoma of the stomach、gastric mucinous cancer and papillary carcinoma of the stomach。Immunohistochemical staining results suggest that45cases of TSP50protein ispositive, the positive rate is88%。We also gather5cases of fresh gastric cancerspecimen，RT-PCR results display that4cases of TSP50mRNA is positive, thepositive rate is80%。From the level of protein and mRNA proofed that TSP50highly express in gastric cancer，it conforms to characteristic that TSP50is a cancer-testis antigen。2、expression mechanism of TSP50in gastric cancerApplied to the methylated sites sequencing to detect methylation pattern of CpGsites in promoter in gastric cancer with TSP50was positive and gastric cancer withTSP50was negative，respectively。The methylation level of CpG sites in promoter ingastric cancer with TSP50is positive is0-54%，the methylation level of CpG sites inpromoter in gastric cancer with TSP50is negative is67%-100%。Furthermore，thereare36methylated sites in this promoter，36methylated sites are at high levelmethylation in gastric cancer with TSP50is negative。However，in gastric cancer with TSP50is positive，there are21methylated sites which are at low level methylation，even totally demethylated。The analysis speculate that these21methylated sites relateto activity of promoter，and play a important role in regulate expression of TSP50。To sum up，when TSP50is negative，CpG sites in it’s promoter are at high levelmethylation，while TSP50is positive，CpG sites in it’s promoter are at low levelmethylation。Thus，the methylation level of CpG sites in TSP50promoter play aimportant role in regulate expression of TSP50。...