The Expression Of DNA Methyltransferases And The Genomic DNA Methylation Pattern In Gastric Cancer

Epigenetics is the study of inherited genetic changes that occurwithout changes to the DNA sequence. The mechanisms of epigeneticchange include DNA methylation—the most widely researched epigeneticalteration in human tumor cells—histone modification, and chromatinremodeling. Human tumor cells exhibit aberrant DNA methylation patternsincluding the hypermethylation of CpG islands in tumor-suppressor genes(TSGs) and a global loss of DNA methylation in the genome. Thesechanges, which are related to the inactivation of TSGs and the activation ofoncogenes or tumor-promoter genes (TPGs), may promote tumorprogression. Abnormal expressions of DNA methyltransferase (DNMT)may play important roles in the aberrant DNA methylation that occurs intumors. DNMT expression can be significantly higher in tumors than incontrol tissues, and the over-expression of DNMT may contribute to tumorprogression through the hypermethylation-mediated inactivation of TSGsin CpG islands. Because DNMT inhibitors can reverse themethylation-dependent TSG silencing,5-aza-2’-deoxycytidine (a DNMT inhibitor) should be useful for tumor treatment. In this study, we usedgastric cancer (GC) to analyze the expressions of DNMTs (DNMT1,DNMT2, DNMT3A, DNMT3B, and DNMT3L) and the effect of5-aza-2’-deoxycytidine on cell inhibition. And we studied the aberrantDNA methylation pattern of GC genome. We expect that this research willbe helpful for the future Epigenetics studies on GC.CHAPTER1The Expressions of DNMT Proteins in GastricCancer and Matched Mucosa TissuesObjective: To investigate the protein expressions of DNAmethyltransferases (DNMTs, including DNMT1, DNMT2, DNMT3A,DNMT3B and DNMT3L) in gastric cancer (GC) and matched gastricmucosa tissues.Methods: Immunohistochemistry was performed to detect the DNMTexpressions in30pairs of GC and matched mucosa tissues.Results: GC and matched mucosa tissues expressed all five kinds ofDNMT proteins. The proliferating zones of foveolar epithelia in gastricmucosa tissues expressed DNMT proteins significantly. The expression ofDNMT3A was weaker in GC tissues than in matched controls. Theexpressions of other DNMTs, however, did not differ significantly betweenthe GCs and controls. In GC tissues, the positive rates of DNMTexpressions (with the exception of DNMT2) were higher in diffuse type than in intestinal type.Conclusion: GC did not necessarily show stronger expressions ofDNMT proteins than the matched mucosa tissues. The DNMT expressionsin GC were associated with the Lauren’s type.CHAPTER2The Distributions of DNMT Proteins in Gastriccancer and the effect of5-aza-2’-deoxycytidine on Gastric cancercellsObjective: To investigate the protein expressions and distributions ofDNMTs in GC and gastric epithelial cells and to analyze the effect of5-aza-2’-deoxycytidine on GC’s growth rates, cell cycle distributions andapoptosis rates.Methods: Immunofluorescence and western blotting were performedto detect the DNMT distributions and expressions in GC cells (SGC-7901and MKN-28) and gastric epithelial cell (GES-1). MTT assay and flowcytometry were performed to analyze the reactions of GC cells to5-aza-2’-deoxycytidine treatment.Results: SGC-7901and MKN-28expressed5kinds of DNMTproteins. DNMT1, DNMT2and DNMT3L were distributed in the nuclei ofGC cells. DNMT3A and DNMT3B were distributed in both the nuclei andcytoplasm of GC cells. The DNMT expressions did not differ significantlybetween the GC cells (SGC-7901, MKN-28) and gastric epithelial cell (GES-1).5-Aza-2’-deoxycytidine with concentrations of0.5-5μmol/L(where it acts as a demethylation drug) had very little effect on the growthrates, cell cycle distributions, and apoptosis rates of SGC-7901or MKN-28cells when the drug was used alone in short term (5days).Conclusion: GC cells didn’t necessarily express DNMT proteinsstronger than gastric epithelial cells.5-aza-2’-deoxycytidine (0.5-5μmol/L)didn’t necessarily inhibit GC cell proliferation when it was used alone inshort term.CHAPTER3The aberrant DNA methylation pattern of Gastriccancer genomeObjective: To investigate the aberrant DNA methylation pattern of GCgenome.Methods: Methylated DNA immunoprecipitation (MeDIP) microarray(MeDIP-chip) was performed to analyze the genomic DNA methylation ofa pair of GC and matched mucosa tissue. MeDIP-qRCR assay wasperformed to validate parts of the MeDIP-chip findings in5pairs of gastrictissues. qRT-PCR was performed to analyze the mRNA expressions ofvalidated genes (such as ABL2, FGF18, TRAF2, EGFL7and RAB33A) inthe5pairs of gastric tissues above.Results: For both the GC and matched mucosa tissue, localhypermethylation and global hypomethylation of genomic DNA were the significant characteristics. And certain CpG islands and gene promoterswere hypermethylated only in GC tissue, whereas some other CpG islandsand gene promoters were hypermethylated only in matched mucosa tissue.After separating tumor related genes those showed differential DNAmethylation in promoters between the cancer and control from the genome,GC tissue was found to contain both aberrantly hypermethylated andhypomethylated TSGs, oncogenes, and tumor-promoter genes (TPGs).MeDIP-qPCR verified some tumor related genes (such as ABL2, FGF18,TRAF2, EGFL7and RAB33A) were aberrantly hypomethylated in GCtissues compared to in controls. And the qRT-PCR assay found that themRNA transcriptions of FGF18, TRAF2and EGFL7were significantlymore in GC tissues than in matched mucosa tissues, but the mRNAtranscriptions of ABL2and RAB33A were significantly less in GC than inmatched controls.Conclusion: Aberrant DNA methylation pattern of GC genome iscomplex, but not limited to hypermethylation of TSG only.