| BackgroundGastric cancer is one of the most common malignant tumors in our country.The morbidity and mortality of gastric cancer rank fourth and third in the world,respectively.In recent years,studies have reported that the occurrence and development of gastric cancer was a multi-factor and multistage lesion process with abnormal expression and accumulation of multiple genes.In addition to the classical gene deletion and mutation mechanism,epigenetics has attracted much attention in the development of gastric cancer.The gene promoter methylation may be the third important mechanisms of gene expression regulation.Tumor suppressor gene(TSG)is one of the negative regulatory signals in the process of cell proliferation,differentiation and apoptosis.Gene inactivation caused by high promoter methylation of tumor suppressor gene contributes to the unlimited growth of cells and promotes the formation of tumor.Therefore,it is helpful to explain the process of cell proliferation,differentiation,and carcinogenesis.As abnormal methylation of tumor suppressor gene can be detected in the early stage of tumor,by screening the methylation profile of gastric cancer,it can provide theoretical and experimental evidence for early diagnosis,treatment and prognosis.ObjectiveThrough analyzing the methylation status of the promoter region of tumor suppressor genesp16 hMLHl and CDH1 and their protein expression levels in gastric cancer tissues,the correlation between them becomes explicit.The role of these 3 genes in the occurrence and development of gastric cancer can be revealed by statistically analyzing the association between the methylation status of the promoter region and protein expression levels of these 3 genes and the clinical pathological parameters and prognostic parameters.Through the cell function experiment,we preliminary study the tumor suppressor mechanism of over expression of tumor suppressor gene hMLHl in gastric cancer,which is in gastric cancer cell line and normal gastric mucosal epithelial cell line.Methods1.Detection of p16,hMLHl and CDH1 gene promoter methylation in gastric cancer tissues and its correlation with clinicopathological features and prognosis We randomly selected 120 patients diagnosed by pathology with primary gastric cancer and without being treated with radiotherapy and chemotherapy before operation.Tumor tissues and paired normal tissues adjacent to cancer 5cmwere collected after radical operation.QIAamp DNA Mini Kit was used to extract DNA,followed by the hydrogen sulfite conversion.Methylation-specific PCR(MSP)was also used to detect the methylation level of CpG Island in p16,hMLHl and CDH1 genes promoter region.Chi square test or Fisher exact test in SPSS 18 statistical software were used to analyze the correlation between the methylation level and clinical parameters of each gene.The relation between p16,hMLHl and CDH1 gene methylation status and the overall survival of patients was discussed by the Kaplan-Meier method.Log-rank test was adopted on overall comparison and survival curve was drawn.2.Detection ofthe expression levels of p16,hMLHl and E-cadherin in gastric cancer tissues and their correlation with clinicopathological features and prognosis RoutineHematoxylin-eosin staining(HE)and Immunohistochemical staining(IHC)were performed on 120 gastric cancer tissues and adjacent tissues.All steps were carried out according to the product specification and PBS(0.01mol/L,pH7.4)was used instead of one anti as negative control.Results of immunohistochemical staining:pl6and E-cadherin protein positive staining signals were localized in the cytoplasm while hMLHl protein positive staining signal was localized in the nucleus,which acted as brownish yellow granules.Each specimen was observed with 2 high power field representative and each field 100 cells were counted to determine the percentage of positive cells.Then we took their average value.The number of positive cells less than 20%was negative(-)and the number of positive cells more than 20%was positive(+).Chi square test or Fisher exact test in SPSS 18 statistical software were used to analyze the correlation between the methylation level and clinical parameters of each gene.The relation between p16,hMLH1 and E-cadherin protein expression levels and the overall survival of patients was discussed by the Kaplan-Meier method.Log-rank test was adopted on overall comparison and survival curve was drawn.The relation between gene promoter methylation status and protein expression was analyzed by Spearman correlation analysis.3.Detection of methylation of hMLHl gene promoter region and mRNA expression level in gastric cancer cell line and normal gastric mucosa cell line Gastric cancer cell lines(MKN-74,MKN-45,SGC-7901,HGC-27,MGC-803,BGC-823 and AGS)and normal gastric mucosal epithelial cell line GES-1 were cultured in vitro.The cells were inoculated in the DMEM/F12 culture medium containing 10ml/L fetal bovine serum(inactivated under 56℃ for 30min)and cultured in a constant temperature closed type incubator with 5%CO2 under 37 ℃.During the logarithmic growth phase,1*106 cells were performed on DNA extraction and heavy hydrogen sulfite conversion.The methylation level of the promoter of hMLH1 gene was detected by MSP in 8 cell lines.Trizol reagent was used to extract the total RNA,and the reverse transcription was synthesized to cDNA according to the specification of RT-PCR kit.Quantitative PCR amplification was used to analyze the relative expression level of mRNA gene hMLH1 in each cell line.Gastric cancer cell line which had high methylation of promoter region and decreased expression of mRNA was obtained.4.Clone formation assay and Transwell test Lentiviral vector system with wild type was structured.Gastric cancer cell line AGS with high methylation and decreased expression of mRNA in the promoter region was transfected.Stable expression of exogenous hMLH1 and negative control AGS cell lines were established.The effect of hMLH1 overexpression on the ability of AGS cells clone formation was detected by the clone formation assay.And the effect of hMLHl overexpression on the invasion ability of AGS cells was analyzed by the Transwell assay.Results1.MSP detection results showed that the positive rates of methylation of p16,hMLH1 and CDH1 genes promoter region in 120 cases of gastric cancer were:36.67%(44/120),24.17%(29/120)and 25.00%(30/120).In adjacent tissues,the positive rates of methylation of p16,hMLH1 and CDH1 genes promoter region were 25.83%(31/120),8.33%(10/120)and 14.17%(17/120).The positive rates of methylation of hMLH1 and CDH1 genes in gastric cancer tissues and adjacent tissues were statistically significant(p<0.05).2.The methylation status of hMLH1 gene was related to alcohol(p = 0.019),CA199 level(p<0.001)and Borrman(p = 0.030).The rate of hMLH1 methylation was higher in the patients with drinking history(40%)than in the patients without drinking history(18.9%).The hMLH1 methylation rate in gastric cancer patients whose CA199 was more than 37kU/L(53.57%)was higher than that in those CA199 was less than or equal to 37kU/L(15.22%).The hMLH1 methylation rate was higher in patients with Borrman type I and type II(66.67%)than in patients with type III and type Ⅳ(21.93%).The methylation status of CDH1 gene was related to the level of CEA(p = 0.003).The CDH1 methylation rate was higher in gastric cancer patients whose CEA was more than 5 g/L(46.43%)than that in those whose CEA was less than or equal to 5 g/L(18.48%).Thus methylation of hMLHl gene promoter is associated with overall survival in patients with gastric cancer,which is an independent risk factor for predicting prognosis.And gastric cancer patients with high methylation of hMLHl gene have a worse prognosis.The methylation status of p16 and CDH1 gene was not related to the overall survival of patients with gastric cancer.In the joint analysis,the cancer patients with 0 or 1 gene methylation in cancer tissues had a better prognosis than those with 3 or 2 gene methylation(χ~2 = 3.901,p= 0.048).3.The results of immunohistochemistry showed that the positive rates of p16,hMLH1 and E-cadherin protein expression in 120 cases of gastric cancer were:36.67%(44/120),69.17%(83/120)and 68.33%(82/120).In the adjacent tissues,the positive rates of p16,hMLHl and E-cadherin protein expression were:86.67%(104/120),94.17%(113/120)and 92.50%(111/120).The positive rates of p16,hMLH1 and E-cadherin protein expression in gastric cancer tissues and adjacent tissues were statistically significant(p<0.05).4.The expression of p16 protein was related to tumor size(p = 0.030).The positive expression rate of p16 was higher in tumor patients with mass diameter<6cm(44.44%)than patients with mass diameter of the tumor ≥ 6cm(25.00%).The expression of hMLHl protein was related to the level of tumor marker CA199(p =0.041)and tumor size(p = 0.036).The positive rate of hMLHl expression was lower in gastric cancer patients whose CA199 was more than 37kU/L(53.57%)than that in those whose CA199 was less than or equal to37kU/L(73.91%).The positive expression rate of hMLHl was higher in tumor patients with mass diameter<6cm(76.39%)than patients with mass diameter of the tumor ≥ 6cm(58.33%).E-cadherin protein expression was associated with alcohol consumption(p = 0.041).The positive rate of E-cadherin expression in gastric cancer patients with drinking history(53.33%)was lower than that in the patients without drinking history(73.33%).The expression level of p16,hMLHl and E-cadherin protein was not related to the overall survival of patients with gastric cancer.There was a negative correlation between hMLHl gene methylation and its protein expression in gastric cancer(p = 0.005).5.The promoter of hMLHl gene in gastric cancer cell lines(MKN-74,MKN-45,SGC-7901,HGC-27,MGC-803,BGC-823,and AGS)was methylated.The mRNA expression levels of hMLHl in all gastric cancer cell lines were lower than those in normal gastric mucosa.AGS cell group infected by the over expressed virus of MLH1 gene was labeled as the experimental group,and AGS cell group infected by the negative control virus was marked as the control group.The results of clone formation test showed that the number of clones in the control group and the experimental group were 180 ± 3 and 169 ± 6,and the number of clones in the experimental group was higher than that in the control group(p = 0.039).The results of Transwell test showed that the number of cells in the control group and the experimental group were 135 ± 4.08 and 125 ± 4.18.The number of transmembrane cells in the experimental group was higher than that in the control group(p = 0.048).ConclusionAberrant methylation of p16,hMLH1,and CDH1 genes in gastric cancer is a frequent event.The abnormal methylation of CpG island in the promoter region of multiple tumor suppressor genes is one of the important mechanisms of gastric cancer.Methylation of hMLHl and CDH1 genes promoter region may be an important molecular event in the carcinogenesis of gastric cancer,which can be used as a potential molecular marker for early diagnosis of gastric cancer.Promoter methylation of hMLHl gene can promote the formation and invasion of gastric cancer cells through down regulating of gene expression,thus to participate in the occurrence and development of gastric cancer.hMLHl promoter methylation is associated with overall survival in patients with gastric cancer,and it is an independent risk factor for postoperative prognosis in patients with gastric cancer.Meanwhile,the methylation of multiple genes is also associated with the prognosis,which can provide theoretical basis for accurate treatment and prognosis for gastric cancer patients. |
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