Exploration Of Sensitive DNA Methylation Markers In A Gastric Cancer High-Risk Region Of China

Objective:Gastric cancer (GC) is one of the commonest gastric-intestinal malignancies in China, it is the second commonest cause of cancer-related death worldwide, and its incidence is particularly high in East Asia, Eastern Europe, and parts of Central and South America. The development of gastric cancer (GC) is a stepwise course accompanied with multiple genetic and epigenetic alterations. Most of the patients lost the chances to have surgical therapy, because the patients didn't have obviously syndromes in their early stages, and the sensitive methods to diagnosis GC in the early stage were insufficient. So it's necessary to explorate the genetic alterations closely linked with GC formation and dissemination would be of great values in prevention, early detection and prognostic evaluation of this malignancy.The development of GC including two fractions: alteration of genetics and epigenetics. Recent studies suggested that epigenetic modifications were more important in gastrocarcinogenesis. Epigenetics refers to changes in pheotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence. DNA methylation plays an essential role in several epigenetic phenomena, including genome imprint- ting, X-chromosone inactivation, and retroelement silencing. DNA methyla- tion was the most important modification in epigenetics, it was catalyzed by DNA methyltransferases, a family of enzymes that comprises DNMT1, DNMT2, DNMT3A and DNMT3B in human cells. CpG dinucleotides can be clustered in CpG islands, which are often associated with promoter regions and remain unmethylated for most genes, the size of CpG islands were 300-3000bp and most of them located in the promoter regions.Dalian belongs to GC high-risk region in China. Its special geo- graphical and physiognomy characteristics (a coastal and mountainous area located at the far south of Northeast China), ecological environment, the particular diet habits of its native residents, and relatively close genetic backgrounds play key roles in the development of GC. Epidemiologic studies have demonstrated that people in this area were accustomed to eating toasted or salted seafood and meat. These foods are rich in gastro- carcinogenesis such as heterocyclic amines, polycyclic aromatic hydrocar- bons, and the well-known DNA methylators such as nitrite and benzo(a)- pyrene which may enhance the risk of genetic and epigenetic alterations, so we should pay more attention to environmental and dietary factors.Aberrant hypermethylation in promoter regions thought to be associated with silencing of some genes, now more and more genes were found to be inactived by DNA methylation, specifically tumor suppressor genes and differentiation-associated genes. Based on the rationale that hypermethylation-induced gene silencing could be uncovered by gene demethylation and reactivation, many lab have analyzed gene expression patterns in human cancer cells with experimentally reduced DNA methyl- lation levels. So DNA methyltransferase inhibitors, like 5-aza- cytidine, 5-aza-2'-deoxycytidine and, to a lesser extent, zebularine, have found wide- spread use. Because of the strong correlation between promoter hyper- methylation and gene silencing, demethylating drugs have been extensively used for the identification of epigenetically silenced cancer genes.In 1999, two distinct subgroups of colorectal cancers were identified that displayed low and high levels of tumor-specific methylation, respec- tively. The latter group of tumors was referred to as exhibiting a"CpG island methylator phenotype"(CIMP). The CIMP group was defined as having concordant tumor specific DNA methylation and could be clearly distinguished due to exhibiting higher methylation index in comparison to non-CIMP tumor-specific methylation. CIMP has been reported in several other tumor types such as gastric, lung, liver, ovarian and leukaemias. We tried to establish a new CIMP system which was suitable for prevention and early diagnosis of GC.Our stydy aims at exploring the role of epigenetic in gastrocarcino- genesis and the relationship between hypermethylation in promoter regions and gene expression silencing through testing epigenetic changes in GC tissues, premalignant tissues, noncancerous mucosa and normal gastric mucosa, which can provides some new evidences and ideas for prevention and early diagnosis of GC in clinical aspect.Methods:All specimens in this study were selected from the Human Frozen Gastric Tissue Bank of the Cancer Institute, Liaoning Laboratory of Cancer Genomics, Dalian Medical University. We tested protein and mRNA expression of p16, hMLH1, TIMP3, DKK3 and E-cadherin in gastric cancer tissues and four gastric cancer cell lines , we also tested 10 loci methylation statuses in different gastric lesions and gastric cancer cell lines by using immunohistochemistry, immunocytochemistry, RT-PCR, MSP, Western- blotting, cell culture and DNA methyltransferase inhibitors. We tried to establish a new CIMP system to help early diagnosis of GC in Dalian region. The data were statistically analyzed by Kruskal-Wallis and Mamm Whitney with SPSS12.0 software.Results:1. Methylation of the 10 loci in gastric tissuesThe methylation statuses of 10 loci (p16, hMLH1, TIMP3, RASSF1A, DKK3, THBS1, MINT1, MINT2, MINT31 and E-cadherin) in different gastric samples were examined by methylation-specific PCR. The samples checked were 24 gastro-endoscopic samples with mild to moderate inflammation of GC-free patients, and 21 tumor-adjacent noncancerous mucosa, 18 premalignant lesions (13 atrophic gastritis and 5 intestinal metaplasia) and 33 GC tissues (16 intestinal type GCs and 17 diffuse type GCs) of 33 GC cases. As demonstrated in the methylation frequencies of all 10 loci increased as the progression of GC-related lesions. Of the 10 checked loci, the methylation of MINT1, TIMP3, RASSF1A, DKK3, hMLH1 and, especially, p16 could be detected in endoscopic mucosa in the rates of 8.3%, 8.3%, 8.3%, 12.5%, 12.5 and 42.9%, respectively; the methylation rates of MINT31 and E-cadherin were very low (both 4.2%) and MINT2 and THBS1 were none. Increased methylation frequencies of p16, hMLH1, TIMP3, RASSF1A, DKK3 and MINT2 were found in the tissue samples from GC patients irrespective to the types of the lesions, whereas more frequent methylation of MINT31 (30.3%), MINT1 (42.4%) and E-cadherin (54.5%) were found in GC group. Significant differences of hMLH1, TIMP3, RASSF1A, MINT1, MINT2 and E-cadherin methylation rates were established between the premalignant or GC groups and endoscopic group (p<0.05), and hMLH1, TIMP3, RASSF1A, MINT2 and E-cadherin between the noncancerous group and endoscopic one (p<0.05).2. Methylation of the 10 loci in four gastric cancer cell linesThe methylation patterns in the four GC cell lines were variable in the form of 2 methylated loci (p16 and TIMP3) in AGS cells, 3 loci (TIMP3, RASSF1A and MINT1) in MGC803 cells, 4 loci (TIMP3, RASSF1A, MINT1 and MINT2) in BGC823 cells and 5 loci (p16, TIMP3, RASSF1A, MINT2 and E-cadherin) in HGC-27 cells. According the literature, MGC803 and BGC823 were derived from well differentiated Chinese adenocarcinomas, AGS from moderately differentiated Ameracan adenocarcinoma and HGC-27 line from a Janpanese patient with undifferentiated gastric cancer.3. Expression levels of five genes in gastric lesion tissues and GC cell linesp16, hMLH1, TIMP , DKK3 and E-cadherin have important effects in gastrocarcinogenesis, these five parameters were chosen for case-by-case correlative analysis of their methylation with expression by the methods of TMA-based immunohistochemical staining. The expression of p16, hMLH1, TIMP3, RASSF1A, DKK3 and E-cadherin in four GC cell lines was examined by RT-PCR as well. The results revealed that p16, hMLH1, TIMP3, DKK3 and E-cadherin could be detected in all endoscopic mucosa, whereas their expression frequencies decreased to 50.0%, 15.0%, 50.0%, 38.5% and 50.0% in GC samples, respectively. Moreover, the rates of p16,hMLH1 and E-cadherin expression were significantly different between endoscopic group and noncancerous one (p<0.05). Interestingly, aberrant methylation of TIMP3 was detected in the four GC cell lines, but immunocytochemical staining and RT-PCR showed down-regulated but not absent TIMP3 expression in MGC803, BGC823 and HGC-27 cells, indi- cating potential incomplete methylation of this gene or limited effect of methylation on suppressing TIMP3 expression in those cells.4. Establish of CIMP systemp16, hMLH1, TIMP3, RASSF1A and MINT2 were chosen here for profiling CIMP patterns of Dalian GCs, their premalignant and non- cancerous counterparts and the endoscopic gastric mucosa from GC-free patients. Among the 24 endoscopic gastric biopsies, 9 cases (37.5%) were found with p16 methylation, three (12.5%) with hMLH1, two (8.3%) with TIMP3 and RASSF1A, and none with MINT2 methylation (Table 3). According to Toyota's criteria of CIMP classification, none of those sam- ples exhibited CIMP-H, whereas 13/24 (54.2%) were found with CIMP-L. Interestingly, the types of methylated loci (hMLH1, TIMP3, RASSF1A and MINT2) in this group were similar with the ones obtained from GC patients although their frequencies were significantly lower than the later ones (p< 0.05).5. The use of DNA methyltransferase inhibitorIn order to confirm the responsibility of hypermethylation for the loss of p16, RASSF1A, TIMP3 and E-cadherin expression, 5-Aza-dC, an inhibitor of eukaryotic DNA methyltransferase, was used to treat HGC-27 cells with p16, RASSF1A, TIMP3 and E-cadherin methylation and MGC803 cell with RASSF1A methylation.Conclusions:1. Epigenetic modification is an important event during carcinogenesis and Dalian is a GC high-risk region and showed high methylation fre- quencies of multiple loci. The numbers of methylated genes were increased and the methylated levels increased with the progression of gastric carcinoma. Results showed that the methylation status of Dalian was more sensitive than other regions in early stage of GC, this may correlated with dietary and environmental conditions of Dalian.2. CIMP system was used to evaluated the methylated status of whole genome, individual CIMP system was important for predicting and preventing tumorigenesis. We found that different regions have different methylated status, therefore, we need test more GC sensitive genes and chose the most suitable genes to establish CIMP system. We used five genes (p16, hMLH1, TIMP3, RASSF1A and MINT2) to compose a new CIMP system which showed high frequence in the early stage of GC.Aberrant hypermethylation of tumor suppressor genes and repair genes in the promoter CpG island is known to inhibit transcriptional initiation and cause permanent silencing of the genes, which play a crucial role in carcino- genesis. The results improved this, almost all the genes we test have negative correlation between expression and methylation status.3. TIMP3 gene maybe exist incomplete methylation.4. Base on the rationale that hypermethylation-induced gene silencing could be uncovered by gene demethylation, and the methyltransferase inhibitors shown significant clinical benefits, recent analyses revealed substantial drug-induced methylation changes that can now be used as endpoints for the further refinement of clinical treatment schedules. Futher optimization of epigenetic cancer therapies should be feasible through the use of novel DNA methyltransferase inhibitors with improved specificity.