| Human hepatocellular carcinoma is one of common fatal cancers and has poor prognosis. It seriously threaten human healthc,so deeply studying on the development of hepatocellular carcinoma and strengthening the liver cancer prevention have great significance.With the development of molecular biology,we have gradually come to realize that epigenetic regulation and transcriptional regulation of cancer gene are closely related. This adjustment does not change epigenetic gene coding sequence and is reversible, so the scientis pay more attention to this adjustment. Research shows that epigenetic and / or genetic changes may cause cancer. This is a multi-tumor gene function in the multi-step process. The inactivation of tumor suppressor gene function is necessary for the malignant transformation of cells. DNA methylation which is catalyzed by various methyltransferase enzymes (DNMTs)is a natural DNA modification method, is one of the main content of epigenetics. DNA methylation play an important role in many aspects such as regulating gene expression, regulating embryonic development, genomic imprinting, X chromosome inactivation and the incidence of tumors.In DNMTs family, DNMT3b is the most important one. DNMT3b expressed in the Undifferentiated embryonic stem cells catalyzes the DNA methylation, but in normal cells the expression is very low. CpG islands are recognized as an important regulatory mechanism for the gene expression. About 50% of CpG islands are located in promoterregions. Most of promoter-associated CpG islands are unmethylated in normal cells.The switching from unmethylated to methylated CpG islands is recognized as an essential contributor to gene silencing which associated with a loss of gene function .This process is catalyzed by DNMT3b and eventually lead to the occurrence of cancer. Our study also found that in the process of malignant transformation of hepatic cells, the expression of FHIT gene (an important tumor suppressor gene) is significantly inhibited. Using siRNA to inhibit the expression of the DNMT3b, the FHIT gene's expression is also restored. But in this process,the CpG island of FHIT gene's promoter region was not methylated. The results suggest that in the process of malignant transformation of hepatic cells DNMT3b not only play the role of DNA methyltransferase, but also play the role of transcriptional regulation factors. In fact, DNMT3b has PWWP and PHD domains. These domains are relevant with the protein-protein interactions. DNMT3b have had the basis of signal transduction molecules. Previous studies focused on DNMT3b as enzyme, but its function as a transcriptional regulatory factor has not been reported until now.Signal transducers and activators of transcription (STATs) are the important nuclear transcription factor. STATs family have many members. They play an important role, such as the signal transduction of many cytokines and growth factors and in the regulation of human immune response inflammatory response and cell growth, differentiation, apoptosis. STATs also have the functions of correlation with the growth, proliferation and evolution in human cancers. It also has been reported that the expression of STATs and its downstream gene such as Bcl-2, Cyclin D1, c-myc, VEGF, P21 are abnormal in hepatocellular carcinoma tissue, but the CpG island of STATs gene promoter region are invariably. Researches show that STATs and the downstream genes are great signficance in the development process in tumors. Bcl-2, Cyclin D1, c-myc and VEGF are very important cancer genes. They are great significance in many aspects of the hepatocellular carcinoma such as proliferation, differentiation, apoptosis, angiogenesis, invasion and metastasis and other functions.If we can certificate DNMT3b is associated with STATs signal transduction pathway, this suggested that DNMT3b-STATs signaling pathway is the existence and DNMT3b not only can be used as DNA methyltransferase to catalyze methylation of the gene, affect gene expression but also can play a role as a transcriptional regulatory factor. This will further reveals the diversity of the function of DNMT3b and gives us a new target for cancer gene therapy and provide new ideas for the prevention of hepatocellular carcinoma.Methods:1. Culture SMMC7721 cell line;2. SMMC7721were transfected with DNMT3bsiRNA transfection of hepatoma cells, the final concentration is 50nM;3. After transfected with or without DNMT3bsiRNA at 48h, collection transfected cells and untransfected cells and then cytoplasmic extract the total protein. Western blotting detection DNMT3b expression changes;4. Transfected with or without DNMT3bsiRNA before and after 24 or 48h, Use cell counting plate to count transfected cells and untransfected cell;5. Using Western blotting to detect the expression of STATs and its downstream genes,such as VEGF, Cyclin D1 expression, etc;6. Dealing with the data and coming to a conclusion.Results:1. After transfection with the DNMT3bsiRNA 48h, we use western blotting to detect the expression of DNMT3b in SMMC7721 cell line.we found that DNMT3b expression in transfected cells was significantly lower than untransfected cells and normal cells.2. Compared with the untransfected cells, the growth rate of transfected cells were inhibited. The difference was significant (P <0.05), while there are a large number of dead cells.3. Compared with the untransfected cells, the expression levels of STAT1, STAT3, STAT5 and its downstream Cyclin D1, c-myc, VEGF gene were also decreased ikin the transfected cells.Conclusion:1. The establishment of DNMT3bsiRNA can make us easily study the function of DNMT3b. 2. Finding that the signal transduction pathways DNMT3b-STATs exist in SMMC7721 cell line. Down regulation the expression of DNMT3b can inhibit the expression of STAT1, STAT3, STAT5 and theirs downstream gene Cyclin D1, c-myc, VEGF.3. Finding that down regulation the expression of DNMT3b, can induce the changes in the expression of Cyclin D1 protein, make tumor cells stagnated in G1 phase and inhibit the cell proliferation and apoptosis in hepatocellular carcinoma.
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