| Background: Pancreatic cancer (PC) is one of the most malignant tumors and most patients suffering from pancreatic cancer would die within one year after diagnosis. Now surgical resection is the best choice to treat pancreatic cancer. But the tumor has been in advanced stage or even unresectable when more than 80% of patients were diagnosed pancreatic cancer. This is the reason why the removal rate (4%-27%) and survival rate are so low. In the patients underwent radical surgical treatment, the 5-year survival rate is only 0.4%-4%, the lowest one of all cancers. And the incidence of pancreatic cancer showed an increasing trend year after year epidemiologically. Therefore, early detection, early diagnosis and early treatment are very essential to improve the prognosis of patients with pancreatic cancer. In the current clinical examination, serum CA19-9 has become the best indicator to detect pancreatic cancer due to the high sensitivity (84.9%-86%) and the high specificity (69.7%-73%). However, when the elevated CA19-9 was discovered, the diameter of tumor should be usually more than 3 cm, which limits its role in finding pancreatic cancer in early stage. Others such as serum CA50, CT, MRI examinations are also unsatisfactory in early diagnosis of pancreatic cancer .Different with genetic, epigenetic is a subject to study the changes in gene expression induced not by changes of gene sequence. The research of epigenetic has already well-developed to the epigenome level. Epigenetic changes in cancer research has become one of the most important molecular events. Epigenetic includes DNA methylation, histone post-translational modifications, histone variation, chromatin remodeling (structural changes), genetic imprinting, as well as RNA interference ( non-coding RNA, or gene silencing) and so on. At present, the mechanism of DNA methylation leading to transcriptional inactivation is definite. But the causes of change of DNA methylation in malignant tumors have not yet entirely clear. Different malignant tumors may have the different methylation profile. And we know nothing about the changes of methylation profile in the same tumor with different stages such as precancerous lesions, early cancer and advanced cancer. Because the methylation is reversible, it is important to find methylation changes in cancer in early stage and precancerous lesions and to make appropriate treatment measures, which are likely to play an active role for cancer prevention and treatment.Objectives: 1. To screen gene by MeDIP technique in the DNA samples of normal pancreas and PC cell lines; To observe the possible developmental model of TNFRSF10C promoter methylation in normal pancreas, peri-tumor and cancer, in order to find an early diagnostic indicator and a new therapic target for the advanced cancer. 2. To detect the proliferation, apoptosis, TNFRSF10C gene transcription and translation of PC cells intervented by 5-aza-dC (5-aza-2'-deoxycytidine) and/or TSA (trichostatin A); To evaluate the role of aberrant TNFRSF10C methylation in the mechanism of pancreatic cancer.Methods: 1. Methylation chips were applied to gene screen between normal pancreas and PC cell lines and gene TNFRSF10C (tumor necrosis factor receptor superfamily, member 10c) was selected. 2. COBRA (combined bisulfite restriction analysis) and / or BGS (bisulfite genomic sequencing) were used to test the TNFRSF10C methylation status in four PC cells (BxPC-3, CFPAC-1, PANC-1, SW1990), 6 normal pancreas tissue (N), 27 matched peri-tumors (TP) / tumors (T) which are compared with clinical features. 3. Before and after the treatment of 5-aza-dC and / or TSA on cells, the changes of methylation status were detected by BGS; Apoptosis was observed by Tunel assay and FCM(flow cytometry); and TNFRSF10C gene transcription and translation were checked by real-time RT-PCR and Western Blot. Results: 1.TNFRSF10C was selected out as the research object by methylation chips, RASSF1A as a positive control. 2. Different methylation status occurred in different cell lines: BxPC-3(68.84%), CFPAC-1(0%), PANC-1(96.77%), and SW1990(54.97%). In tissues, the percent of methylation of TNFRSF10C in peri-tumor (TP) and tumor (T) were significantly higher than that in normal pancreas tissue(N) (50.71% vs. 5.84%, P<0.01; 47.89% vs. 5.84%, P<0.01) and no difference between TP and T in the various clinical stages (P>0.05). 3. Different degrees of apoptosis in 4 PC cell lines after drug intervention. In the 3 PC cell lines, except BxPC-3, 5-aza-dC's role in inducing apoptosis was greater than TSA's. The degree of methylation was confirmed by BGS; Except CFPAC-1, TNFRSF10C in the other 3 cell lines was re-transcripted and re-expressed in different degrees.Conclusions: 1. Different DNA promoter methylation occurred in different pancreatic cancer which means different mechanism lead to pancreatic cancer. 2. Aberrant TNFRSF10C promoter methylation is an early event in the pathogenesis of most pancreatic cancer, which can be used as an early diagnostic indicator without estimation of course of disease. 3. TNFRSF10C can be used as a new target of early intervention and chemotherapy of pancreatic cancer postoperatively. |
PDF Full Text Request