| Background and ObjectiveHuman hepatocellular carcinoma (HCC) is one of the most common fatal cancers, and it has very poor prognosis.HCC seriously threatens human's health,hence it is very important to explore the carcinogenesis of HCC,which is helpful for the prevention and cure of liver cancer.With the development of medical science, it has recognized that the occurrence of tumor involved Changes in a variety of gene function and a multi-step process. Even though non-genetic factors also play a role in this process or even an important role, but the central role are the inactivated of tumor suppressor genes(TSG), and the activation of oncogenes. So that the mutant cells gain a selective advantage growth, clonal expansion and eventually transformate malignant of cancer.Formerly, studies of TSG were always focused on genetic defects including gene mutation and deletion. But recent studies reveal the relationship between epigenetic alterations and transcriptional control of TSG, and the epigenetic alterations may influence the function of TSG and consequently cause cancer.DNA methylation is defined as the process by which methyl groups are added to cytosines located in the CpG dinucleotide in genomic DNA, and it is one of the most important epigenetic mechanisms. DNA methylation is catalyzed by DNA methyltransferase (DNMT), and the DNMT3b which functions as a de novo DNA methyltransferase is an important member in the DNMT family.DNA methylation is associated with diverse biological processes such as the regulation of imprinted genes, X chromosome Inactivation, and tumor suppressor gene silencing in human cancer. Many studies show that the CpG islands which are protected from methylation on autosomal chromosomes are methylated in diverse cancers, and the methylation modification in the promoter region has important regulatory effects on gene expression, which is associated with transcriptional Inactivation of defined tumor suppressor genes in human cancers.The abnormal histone modification in tumor is a subject of active research, particularly in unusual histone acetylation and deacetylation.Histone acetylation or deacetylation was Catalytic by the histone acetylases(HATs) or Histone deacetylases(HDACs).The dynamic equilibrium of HATs and HDACs controled the chromatin structure and gene expression, and their dysfunction are the essential molecular mechanism in tumor development. Histone acetylation or deacetylation is a reversible dynamic process,the balance of reversible histone acetylation has a crucial role in the maintenance chromatin structure and regulation of gene expression. Many studies have confirmed that histone high or low acetylation play an important role in the occurrence of tumor.Trichostatin A(TSA) is a metabolite of Streptomyces, which is one of the representatives drug in histone deacetylase inhibitors (HDACI).Study shows that the concentration of TSA at nM class could effectively inhibit tumor cell proliferation, LIU Wei-min found that the concentration of TSA at nM class could inhibit Hepatocellular carcinoma cell line SMMC-7721 growth and induced apoptosis. The zinc finger structure in Histone deacetylase inhibitors (HDACI) has a similar lysine residue side chain structure with histone acetylase, could competitive inhibite the activity of histone deacetylase and improve the level of histone acetylation, chang the structure of chromatin,so as to achieve the role of change the transcription and expression level of specific gene,the main biological effects included induction of tumor cell differentiation, cell cycle arrest and apoptosis.E-cad gene(E-cadherin) is located on 16q22.1,the E-cad protein is a transmembrane glycoprotein,whose molecular weight is 120KDa, it distributed in the same side of the middle connection in the epithelial cells, which is the molecular basis that connected between cell-mediated of connection. E-cadherin protein is widely distributed in the embryo and mature organization of epithelial cells,it regulates embryonic development,the formation of organizations,to maintain the structural integrity of organizations, plays a crucial role in cell-to-cell transmission information and cell adhesion. In human epithelial malignancies, the expression of E-cadherin has also been regulated by the methylation of CpG island in promoter region.Most of the studies shows that the loss expression of E-cadherin has related to the hypermethylation of CpG island in CDH1 gene promoter.Studies have shown that Histone deacetylase inhibitors could reverse the loss expression of certain tumor suppressor gene, thereby inhibited a variety of tumor cell growth included Hepatocellular carcinoma.It has Reported that Histone deacetylase inhibitors(TSA) treatment could restore E-cad gene expression which inactivation by hypermethylation of CpG island in promoter region in bladder cancer cell line.In this study,we use TSA treatment human Hepatocellular carcinoma cell line SMMC-7721, to observe the changes of methylation status in CpG island of promoter region and the expression in E-cad gene, and the relationship between this effect and the expression of DNMT3b,to explore the relationship between histone acetylation that caused by TSA treatment and DNA methylation,find the possible mechanism.Metheds1. Cultured human Hepatocellular carcinoma cell line SMMC-7721.2. Different concentrations of TSA treatment the Hepatocellular carcinoma cell line SMMC-7721, to observe the growth of cells at different times.3. Select the appropriate concentration of TSA treatment the Hepatocellular carcinoma cell line SMMC-7721,collect the cells of test group and control group, extrac total protein and genomic DNA.4. Western blot technology and MS-PCR(methylation-specific PCR) detect the expression of DNMT3b, changes of methylation status in promoter region of E-cad in two groups of cells,Separately.5. Deal with the data, draw conclusions.Results1. MTT method was used to Detection of cell growth curve,calculation of inhibition rate.TSA with the concentration of 300nm/L dealed with the SMMC-7721 cell line significantly inhibited cell growth,when the concentration was 1000nm/L,the cell showed significant cytotoxicity. TSA300nm/L as the experimental concentration, TSA300nm / L to deal with SMMC-7721 cells 24h as the experimental group,the same medium without TSA cultured SMMC-7721 cells as negative control group,The cell growth in experimental group was inhibited, Compared with the control group.The cell growth inhibition rate was 21.85%.2. Western blot detect the expression of E-cad.Western blot detection found the expression of E-cad protein was negative in the control group,the expression of E-cad protein was restored in the experimental group.the expression level of 8-actin is similar in Two groups.3. Western blot detect the expression of DNMT3b.Western blot detection found the positive expression of DNMT3b in the control group,the expression level of DNMT3b was reduced in the experimental group. the expression level of 6-actin is similar in Two groups.4. MSP detect the methylation state in the promoter region of E-cad.In Control group,E-cad gene methylation primers amplified aimed fragment (116bp),and the use of non-methylated primers didn't amplified aimed fragment.In Experimental group, E-cad gene methylation primers amplified negative,and the use of non-methylated primers amplified fragment(97bp).It shows that the CpG island in E-cad gene promoter region is methylated in Control group,After TSA treatment,the CpG island in E-cad gene promoter region is unmethylated in the experimental group.Conclusions1. TSA 300nm/L could inhibit the growth of the human Hepatocellular carcinoma cell line SMMC-7721, when the concentration was 1000nm/L,the cell showed significant cytotoxicity.2. The CpG island in E-cad gene promoter region is methylated in Human Hepatocellular carcinoma cell line SMMC-7721,the expression of E-cad gene is negative,TSA could induce the CpG island in E-cad gene promoter region demethylated,and restore the expression of the E-cad gene.3. TSA could reduce the expression of DNMT3b in human Hepatocellular carcinoma cell line SMMC-7721,and the reduced expression of DNMT3b may play a role in the demethylation of TSA. |
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