| BackgroundOvarian cancer is one of the common female genital cancer, and the mortality accounted for the first gynecological cancer, which is a serious threat to women’s lives. Since ovarian cancer has features of insidious onset, easy to transfer and poor prognosis, about 3/4 of the patients is advanced stage when diagnosed. Surgical therapy combined with chemotherapy can prolong patients survival, but mortality and recurrence of ovarian cancer is still high, and five-year survival rate is still low, therefore, an urgent need for a new molecular markers which can be used for early diagnosis and treatment of ovarian cancer in clinic. Finding new ovarian cancer-associated molecules are the focus of the present study.Prognoses and selection of treatment regimens often rely on imaging, biomarkers, staging, and additional histologic analyses. While many cellular changeshave been noted as hallmarks of cancer, it is often not acknowledged that a change in glycosylation of proteins/lipids is also a hallmark of cancer. A number of biomarkers for cancer useful as diagnostic and prognostic indicators, such as CA-125 Tn and Sialyl Tn, are glycoproteins.Amount them, The Tn antigen is a tumor-associated carbohydrate antigen that is not normally expressed in peripheral tissues or blood cells. Expression of this antigen, which is found in a majority of human carcinomas of all types, arises from a blockage in the normal O-glycosylation pathway in which glycans are extended from the common precursor GalNAcα1-O-Ser/Thr(Tn antigen). This precursor is generated in the Golgi apparatus on newly synthesized glycoproteins by a family of polypeptide α-N-acetylgalactosaminyltransferases(ppGalNAcTs) and then extended to the common core 1 O-glycan Galβ1-3GalNAcα1-OSer/ Thr(T antigen) by a single enzyme termed the T-synthase(core 1 β3-galactosyltransferase or C1GalT). Formation of the active form of the T-synthase requires a unique molecular chaperone termed Cosmc, encoded by Cosmc on the X-chromosome(Xq24 in humans, Xc3 in mice). Cosmc resides in the endoplasmic reticulum(ER) and prevents misfolding, aggregation, and proteasome-dependent degradation of newly synthesized T-synthase. Loss of expression of active T-synthase or Cosmc can lead to expression of the Tn antigen, along with its sialylated version Sialyl Tn antigen as observed in several cancers.Currently,the research of tumor-associated carbohydrate antigens in ovarian cancer is still rare. The expression and posision of T-Synthase, T, Tn and S-Tn in ovarian cancer and normal ovarian tissue, and the relationships between chaperones Cosmc gene that a pivotal role they play in the formation of tumors and TCAC are to be study experimentally. Purpose 1. Identify the expression and distribution of T-Synthase, T, Tn and S-Tn antigen in epithelial ovarian carcinoma tissues and normal ovarian tissues; 2. Explicit T-Synthase, T, Tn and S-Tn antigen expression and distribution in ovarian carcinoma cell lines; 3. Clear biological function of Cosmc transcript variants in ovarian cancer cells. Methods 1. Collecting ovarian cancer tissue slices and epithelial ovarian carcinoma fresh tissue, and then detecting the expression of T-Synthase, T, Tn and S-Tn antigen in ovarian cancer tissues and normal ovarian tissues distribution by immunohistochemistry method and Western Blot; 2. Two ovarian cancer cell lines grown on cover glass. Using immunocytochemistry to detect the distribution and expression of T-Synthase, T, Tn and S-Tn antigen in ovarian cancer cells. Using Western Blot methods to detect the expression of T-Synthase, T, Tn and S-Tn antigen in ovarian cancer cell lines; 3. Construction of Cosmc overexpression lentivirus vector. Western Blot method was used for detecting changes of T-Synthase, T, Tn and S-Tn in ovarian cancer cells after Cosmc transfection. The growth and apoptosis of ovarian cancer cells transfected Cosmc were detected by MTT assay and flow cytometry, using transwell assay to analysis the affection of Cosmc overexpression on ovarian cancer cell migration and invasion. Results 1. The expression of T-Synthase, T antigen, Tn, S-Tn in normal ovarian tissues and epithelial ovarian carcinomaUsing immunohistochemistry to detect T-Synthase, T antigen, Tn antigen, S-Tn antigen expression in 60 cases of carcinoma and 20 cases of normal tissue for. The experimental results showed that T-Synthaseis mainly localized in the cytoplasm, and it was significantly lower in ovarian cancer tissues than that in normal tissues. T antigen mainly located in the luminal surface and the peripheral cell surface, and its expression in ovarian cancer tissues was decreased compared with normal tissues. Tn antigen is mainly localized in the cytoplasm, which expressed significantly higher in ovarian cancer tissues than that in normal tissues. S-Tn antigen was mainly located in the cytoplasm, and its expression was increased significantly in ovarian cancer tissues compared with normal tissues.The expression of T antigen, T-Synthase, Tn and S-Tn were detected by Western Blotting in 20 cases of normal ovarian tissue and 48 cases of epithelial ovarian cancer fresh tissues. Experimental results showed that the expression of T antigen and T-Synthasewas significantly lower in ovarian cancer than that in normal tissues, but the expression of Tn and S-Tn in ovarian cancer tissues were increased significantly compared with normal tissues, The results were consistent with the results of immunohistochemistry. 2. Expression of T-Synthase, T, Tn and S-Tn in ovarian cancer cell line A2780 and Skov3Immunocytochemistry was used to detect T-Synthase, T antigen, Tn and S-Tn expression in ovarian cancer cell line A2780 and Skov3. The results showed that T-Synthase, T antigen presenting low expression or no expression in A2780 and Skov3 cells, while Tn and S-Tn antigen present moderate or high expression in Skov3 and A2780 cells and S-Tn has higher positive rate and sensitivity than Tn.Cosmc, T-Synthase, T, Tn and the S-Tn were detected by Western Blotting in ovarian cancer cells Skov3 and A2780. The results showed that Cosmc, T-Synthaseand T antigen expression were not high in ovarian cancer cells, but Tn and S-Tn antigen showed high expression in Skov3 and A2780 cells. The results were consistent with immunocytochemistry experiments. It indicated that reduced T-Synthaseactivity inhibited the expression of T antigen. 3. Cosmc overexpression on the expression of related molecules and its biological functions in ovarian cancer cellsOvarian cancer cells A2780 and Skov3 were cultured and transfected with Cosmc lentivirus vectors. Transfection efficiency was measured under a fluorescence microscope and the transfection efficiency was above 80 %. The expression of Cosmc significantly increased in Cosmc lentivirus group than in the control cells with more than 2-fold increase. We used Western Blot to detect T-Synthase, Tenascin C, S-Tn, T antigens change in cells. The results showed that, after Cosmc overexpression, T-Synthaseand T antigens expression were significantly increased, while Tenascin C and S-Tn antigen expression was suppressed. It indicating Cosmc may promote the formation of T antigen and inhibit the expression of Tenascin C and S-Tn antigen by upregulating T-Synthase.Number of apoptosis cells were detected by flow cytometry. Experimental results showed that the number of apoptotic cells in ovarian cancer Skov3+ and A2780+(Cosmc overexpressed) was significantly increased compared with control cells Skov3-and A2780. Ovary cancer cell growth ability were detected by MTT assay, and the results showed that the growth ability of the cells was significantly decreased after overexpression of Cosmc. This result indicates that Cosmc overexpression promotes the apoptosis of ovarian cancer cells and inhibit its growth.Skov3 and A2780 cells transfected with Cosmc lentivirus vectors were conducted by transwell experiments to detect the impact of Cosmc on ovarian cancer cell invasion ability. Experimental results showed that the cells getting through the membrane were significantly reduced transfected with Cosmc.It indicated that Cosmc overexpression inhibits ovarian cancer cell invasion capability. Conclusion 1. The expression of T-Synthase, T antigen in ovarian cancer tissues were significantly decreased compared with normal ovarian tissues,and low or no expression in ovarian cancer cell lines.The expression of Tn and S-Tn antigen were significantly increased in ovarian cancer tissues compared with normal ovarian tissues, and expressed highly in ovarian cancer cell lines. 2. The overexpression of Cosmc in ovarian cancer cell lines can promote the expression of T-Synthase, T antigen,and inhibit the growth and invasiveness of ovarian cancer cells, and promote apoptosis. |
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