| Our previous study found that cancer-testis antigen OY-TES-1 was highly expressed in liver cancer, restrictively expressed in normal tissues. There was humoral immune response against OY-TES-1 in cancer patients . It is suggested that it may be a promising target antigen . This study will focuse on elucidating the some of biological function of tumor cells with OY-TES-1 expression, understaning the expression of OY-TES-1 in malignant (coloretal cancer) and benign tumor (meningioma), and humoral response in those patients. The results will help to evaluate the feasibility for immunotherapy of OY-TES-1.Part 1 In vitro experimental study of OY-TES-1 expression in cancer cell effecting on its biological functionAIM: To construct the eukaryotic expression vector pEGFP-N1/OY-TES-1 which then transfect stably into hepatocellular carcinoma cell line HepG2. Transfected cells will be observed in vitro for their growth, proliferation and migration.METHODS: Primers were designed according to the sequence of OY-TES-1 gene in human. Recombinant pMAL-C2/OY-TES-1 was used as a template for amplifing OY-TES-1 cDNA coding region. The PCR product was ligated into pEGFP-N1 vector to construct a recombinant pEGFP-N1/OY-TES-1 identified by DNA sequencing. With Fugene HD reagent, pEGFP-N1/OY-TES-1 was transfected into HepG2 cells that are OY-TES-1 negative expression. Positive clones were screened by using G418, which resulted in gain of cells with stable expression of OY-TES-1. HepG2 cells transfected with pEGFP-N1/OY-TES-1, pEGFP-N1 and untransfected cells, respectively, were tested for their function by cell counting, MTT growth test, plate clone forming, cell cycle detecting and wound-healing experiment.RESULTS: Sequencing result by blast search was confirmed that insertion of OY-TES-1 gene was correct. RT-PCR and immunohistochemistry showed that OY-TES-1 expression in both mRNA and protein lever was higher in cells transfected by pEGFP-N1/OY-TES-1 than un-transfected and transfected by pEGFP-N1 vector only. Through the tests of cell counting, MTT growth test, plate clone formatting, cell cycle detecting and wound-healing experiment, it seems that pEGFP-N1/OY-TES-1 transfected cells do not change in growth, proliferation, cell cycle and migration.CONCLUSIONS: pEGFP-N1/OY-TES-1 eukaryotic expression vector was constructed successfully. It seems that OY-TES-1 had no effect on growth, proliferation, cell cycle and migration in hepatocellular carcinoma cell line HepG2. Part 2 Expression of OY-TES-1 in colorectal cancer and meningioma with its clinical significanceAIM: To investigate the expression of OY-TES-1 expression in colorectal cancer and meningioma, and to explore its significance.METHODS: RT-PCR and immunohistochemistry were used to detect the expression level of mRNA and protein of OY-TES-1 gene, respectively, in 24 adenoma tissues, 60 colorectal cancer tissues and adjacent noncancerous tissues as well as 45 meningioma tissues. Statistic analysis was performed combining OY-TES-1expression of and patients'clinico- pathological features.RESULTS: 73.3% of OY-TES-1 was expressed in colorectal cancer tissues, which was significantly higher than that in adjacent noncancerous tissues (55.0%) and adenomas (45.8%) (P <0.05). The meningioma tissues showed 66.7% of OY-TES-1 mRNA expression. There was no OY-TES-1 protein expression in adenoma and non-cancerous tissues of adjacent colorectal cancer. The positive rate of OY-TES-1 protein expression was 43.3% and 40.0% in colorectal cancer tissues and meningioma tissues, respectively. The OY-TES-1 protein expression in colorectal cancer tissues was significantly associated with depth of invasion and degree of differentiation, and not associated with age, gender, histological type, anatomical site, tumor size, lymph node metastasis, distant metastasis, TNM stage and Duke's stages. There were no significantly differences in OY-TES-1 expression with age, sex and histological type in meningioma.CONCLUSIONS: It is suggested that OY-TES-1 protein had a stricter expression than its mRNA. OY-TES-1 protein was mainly expressed in advanced tumors, and its expression may be related to tumor evolution and malignance. With higher OY-TES-1 expression in both colorectal cancer and meningioma and negative OY-TES-1 expression in non-cancerous tissues,OY-TES-1 may be a useful antigen for tumor auxiliary prognosis.Part 3 Seroreactivity against OY-TES-1 in colorectal cancer and meningiomaAIM: To investigate seroreactivity against OY-TES-1 in colorectal cancer and meningioma, and to explore its clinical significance.METHODS: ELISA was used to detect OY-TES-1 serum antibody in 25 colorectal cancer, 31 meningioma patients and 50 normal donors as control. OY-TES-1 serum immunoreactivity and clinical features were statistically analyzed.RESULTS: The OY-TES-1 antibody was detected in 8.0% of colorectal cancer patients'and in 16.3% of meningioma patients'sera, while no serum reactivity was detected in 50 normal donors. There were no significantly differences between OY-TES-1 serum immunoreactivity and clinical features of patients.CONCLUSIONS: Seroactivity against OY-TES-1 presents specifically in patients with colorectal cancer and meningioma. The significance of OY-TES-1 antibody in patients needs to further explore.
|
PDF Full Text Request