The Study Of Expression And Transcription Of Methyl-CpG Binding Domain Protein In Pancreatic Carcinoma

As an important transcription factor, methyl-CpG binding domain protein1 (MBD1) mediated transcription-suppressing course and might cause descentexpression of numerous tumor suppressor genes, or even result in devitalization ofthose genes. The expression and role of MBD1 were studied in experimental andclinical part respectively. Experimental study: MBD1 mRNA expression level was detected in twopancreatic cancer cell lines, AsPC-1 and BxPC-3, by method of RT-PCR. Theexpression of MBD1 was very high in two cell lines. BxPC-3 was cultured frompancreatic carcinoma in situ, while AsPC-1 was cultured from the ascites of a latestage patient. AsPC-1 not only had strong metastasis vitality but also had biologicalcharacter of the primary cancer. We choose AsPC-1 as our further research patform. By RNA interference (RNAi) technology, the siRNA was designed andsynthesied which aimed at the MBD1 gene. BgLⅡand HindⅢ restriction enzymesites were introduced into the 5' and 3'of MBD1 gene siRNAs respectively, theninserted into polylinker site of plasmid Rotro Super, MBD1 siRNAs eukaryoticexpression vector Rotro Super-MBD1 was constructed. MBD1 siRNAs had beensuccessfully integrated into the plasmid. MBD1 siRNAs vector was transfected intopancreatic cancer cell AsPC-1 by liposome. Positive clone was obtained by the screenof G-418. MBD1 expression level was detected by RT-PCR, and MBD1 protein was testedby western-blot, growth curve MTT assay and clony forming test were used to assessthe proliferation potency. Multi-dots gene chip was used to detect the expression levelof methylation related cancer suppressor genes. It was demonstrated that MBD1siRNAs could significantly suppress expression of MBD1 mRNA in AsPC-1, whichwas the same for protein expression. Growth curve and clony forming test show thatcell growth is significantly inhibited. It demonstrates that MBD1 siRNAs can inhibitthe proliferation of pancreatic cancer cells, reduce its survival ability and adaptability.The methylated cancer suppressor genes, such as CDH1, Rb and E2F5 wereup-regulated in the multi-dots gene chip experiment. The expression of MBD1 in pancreatic carcinoma was detected at protein levelby immunohistochemistry, at gene level by RT-PCR respectively. MBD1 expressionwas significantly higher (76.32%) in pancreatic carcinomas than that in normalpancreatic tissue, benign pancreatic tumors, corresponding distant pancreas tissues 6博士论文 第 7 页 共 100 页and chronic pancreatitis measured by immunohistochemistry. The expression ofMBD1 mRNA in pancreatic carcinomas was also obviously higher than normalpancreatic tissues, or the distant pancreatic tissues (P<0.01) tested by RT-PCR. Nocorrelation was found between the expression of MBD1 with the sex, age, location,size, differentiation or the staging of pancreatic carcinoma. MBD1 expressed in92.31% pancreatic carcinomas with lymph node metastasis, which is higher than thatin pancreatic carcinomas without lymph node metastasis (41.67%). Among them, 7samples with MBD1 strongly expressed were testified having para-abdominal aortalymph node metastasis (station 16). The influence of intra-arterial chemotherapy onMBD1 gene expression was also studied. MBD1 gene expression declined afterintra-arterial chemotherapy. Conclusion: the expression level of MBD1 was significantly higher in pancreaticcarcinoma and it might have close correlation with metastasis of pancreatic cancer.MBD1 was one of the oncongenes of pancreatic carcinoma. MBD1 siRNAs couldinhibit the expression of MBD1 and the proliferation of pancreatic cancer cellsAsPC-1, up regulating expression level of other methylated cancer suppressor genes,and MBD1 may be a new target of gene therapy for pancreatic carcinoma. Regionalintra-arterial chemotherapy was an effective treatment by inhibiting the expression ofMBD1.