| Human hepatocellular carcinoma is a fatal tumor. It has a serious threat to human health and has poor prognosis and poor treatment effect. Therefore,deeply study the mechanism, the occurrence and development of hepatocellular carcinoma, which has important significance to strengthen the prevention and treatment of hepatocellular carcinoma.In genome mapping, gene CyclinD1 is located in 11q13, approximately 15KB in length, consisting of 295 amino acid residues encoded protein. The protein molecular weight is about 36kb. The action mechanism of CyclinDl gene is:CyclinDl leads to pRb phosphorylation through the activation of CDK4, releasing of nuclear transcription factor E2F, prompting the copy nuclear DNA, and then the cells from G1 phase to S phase were accelerated. The result is the malignant proliferation of the cell.MiRNAs is a length of about non protein coding small single stranded, endogenous, highly conserved RNA, including 18-25 nucleotide composition. Modern research has found, plants and animals with a variety of miRNAs, the main function is through the inhibition of protein translation or degradation of the target mRNA. They have a close relationship with the occurrence and development of the metabolism of the body, the individual development and so many kinds of diseases which include of the regulation of cell growth, differentiation, apoptosis and proliferation; Research shows that, miRNA can be in a comparatively long time stable exist in human and animal blood, saliva, urine and other body fluids, plays a role in regulation of cell differentiation, development, apoptosis and proliferation. Some of the miRNAs has a role in inhibiting tumor. In tumor tissue miRNAs can promote the formation of certain and evolution of tumor development. Previous research indicates that, miR-145 is a tumor suppressor gene in carcinoma tissue, abnormal expression in hepatocellular carcinoma cell lines, and plays an important role in the occurrence and development of human tumors.In the field of molecular biology research, we have recognized that epigenetic (Epigenetics) change is often caused by transcriptional regulation of tumor gene; we pay more attention to Epigenetics which do not change the gene coding sequence, and the regulation of epigenetics is reversible. A large number of studies show that, the occurrence of tumor is a multiple steps process which composed of multiple tumor genes tumor suppressor gene function change; epigenetic or/and genetic changes also can due to tumor occurrence. The incidence of malignant tumors are generally associated with loss of function of the tumor suppressor gene. Completed by the DNMT3b catalyzed methylation of DNA promoter region is an important modification method of DNA table, is an important content in genetics, such as modified method, gene expression and regulation of X chromosome inactivation, genomic imprinting, embryonic development regulation and tumor incidence.DNMT3b is one of the most important functions of the DNMTs family. In undifferentiated embryonic stem cells, we can detecte a large number of expression of DNMT3b. The mainly function of DNMT3b is catalyze DNA methylation. in normal human cells, DNMT3b expressed very low. DNA methylation regulation is mainly through the methylation state changes of the CPG gene promoter region of the island. The promoter region has about 50% CPG island position. In normal cells, located in the promoter CPG island most sub areas is low methylation status. In the malignant tumor cells, and the high expression of DNMT3b can make the suppressor gene CPG island in the promoter region hypermethylation status in many tumors. The function of hypermethylation is gene silencing which caused the occurrence of tumor.Our study also found in hepatocellular carcinoma in the process of malignant transformation; CyclinDl gene (oncogene important) expression was significantly increased; We use the siRNA tosuppress the DNMT3b expression. CyclinDl gene expression also decreased. In this process, the methylation state of CyclinDl gene is unchanged in the promoter region CpG island.In this process, if the DNMT3b plays a role of methy ltransferase, suppressed DNMT3b expression can enable the CyclinDl gene expression increased, and our experiment found that, in this process, the expression of CyclinDl gene is lower, and the promoter region methylation level unchanged. What is the cause of the CyclinDl gene expression change? This experiment will answer this question.The first partMethods:1, We subculture SMMC7721 cells; the final concentration of 50nM DNMT3b siRNA was transfected into hepatocellular carcinoma cell line SMMC7721;2, Research on packet:The study was divided into experimental group transfected with DNMT3b siRNA, the control group transfected with control siRNA.3, SiRNA was transfected into normal cultured SMMC-7721 cells were inoculated in a 6-well plate, then transfected DNMT3b siRNA.4, With the Japanese company Nikon fluorescence microscopic imaging/ control siRNA camera system for the detection of FITC labeled in SMMC7721 cell transfection efficiency and photography.5, In 48h respectively after transfection of genomic DNA extraction from two groups of cells and Extraction of total protein.6, Using the Western blotting method for detection of DNMT3b and CyclinDl expression in changes before and after transfection.7, Methylation specific PCR (MS-PCR) change detection of methylation status of CyclinDl promoter region before and after transfection of genomic DNA.Results:1, Hepatoma cells were significantly inhibited after transfection. The fluorescein labeled control siRNA, under the same conditions were transfected into SMMC7721 cells, the 37℃ and incubated in 5%CO2 48h culture box, the transfection efficiency was observed by fluorescence microscope, siRNA was transfected into hepatoma cell efficiency can reach more than 90%.2, Using the Western blotting method for detection of DNMT3b and CyclinDl shows the siRNA after transfection, the expression level of DNMT3b and CyclinD1 in experimental group was obviously low, and no significant difference between control group and normal group, the expression level of DNMT3b and CyclinD1.3, By the experiment of MTT, transfection of DNMT3b siRNA in the experiment group and the control group cells proliferation activity at 48 and 72 hours were significantly different.4, Detection of SMMC7721 cells by flow cytometry in experimental group cells transfected with DNMT3b siRNA is the number of cells in G0/G1 phase was higher than that of control group cells, and S phase cells decreased.5, The experimental group and the control group CyclinD1 gene in cell showed no significant difference, the methylation status of M primers, not be amplified, U primers amplified fragment, the methylation of CyclinDl gene in the two groups had no change of state.The second partMethods:1, Cultured in SMMC7721 cells transfected with DNMT3b siRNA2, In 48h respectively after transfection of genomic DNA extraction from two groups of cells and total RNA.3, Before and after transfected DNMT3bsiRNA, using RT-PCR for detection the expression of mir-145 RNA mRNA and CyclinDl mRNA.4, Methylation specific PCR (MS-PCR) change detection of methylation status of mir-145 promoter region before and after transfection of genomic DNA.Results:1, We see that the experimental group mir-145 RNA before transfection low expression after transfection, the high expression of mir-145 RNA; in the control group before and after transfection and expression status of mir-145 RNA invariant, were lower expression. The experimental group and the control group before transfection, the expression of CyclinDl mRNA after transfection were not changed significantly, showed high expression.2, the experimental group and the control group CyclinDl gene in cell showed no significant difference between low methylation, the methylation status of M primers, not be amplified, U primers amplified fragment, the methylation of CyclinD1 gene in the two groups had no change of state.Conclusion:DNMT3b siRNA can specifically inhibit the expression of DNMT3bDNMT3b can regulate the expression of mir-145, CyclinDl gene expression and regulation. The methylation in promoter region of mir-145 gene and CyclinDl gene of the process has not changed. This shows that does not play DNA methyltransferase DNMT3b’s role in this process.The expression of DNMT3b and lead to the low expression of CyclinDl expression induced by high miR-145, the tumor cell arrest in the G1 phase, thus inhibiting the growth and proliferation of hepatocellular carcinoma cells, showed that DNMT3b could be an important molecular target for gene therapy of hepatocellular carcinoma. |
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