Effects Of Helicobacter Pylori Eradication On CpG Island Methylation Status Of E-Cadherin And DAPK Gene In Nonneoplastic Stomach

Purpose To observe the effects of helicobacter pylor(iHp) eradication on CpG island methylation status of E-cadherin and DAPK gene,and the CpG island methylation status and expressions of E-cadherin and DAPK in patients with nonneoplastic gastric mucosal,therefore discuss the possible mechanism of gastric cancer from the genetic and protein level and the meaning of helicobacter pylori eradication on preventing gastric cancer.Methods Collection the endoscopic gastric mucosal tissues of outpatients in our hospital for DNA level detection and histopathology who was Hp infected chronic gastritis by making gastroscope and rapid urease experiment (RUT) check due to indigestion symptoms. Recording the patient's clinical and pathological materials. All Hp infected patients were received standard triple therapy(esomeprazole 20mg,amoxicillin 1.0g,clarithromycin 0.5g, EAC therapy),twice a day for ten days. A carbon-14 urea breath test(14C-UBT)was performed to determine Hp status after the end of the treatment.The patients with successful eradication of Hp were followed up six months prospectively. Selection the chronic gastritis patients who was double positive for the RUT and Warthin-Starry stain methods and had successful eradication of Hp as research objects, double negative for RUT and W-S methods as control. Detection the E-cadherin, DAPK protein expression levels by immunohistochemical method(IHC) and the CpG island methylation status by methylation-specific PCR(MSP).Analysis the association between clinicopathological features and CpG island methylation. Cloning and sequencing methylated products to verify the accuracy and amplification of MSP.Results 1. 67 chronic gastritis patients were double positive for RUT and W-S,41 chronic gastritis patients were double negtive for RUT and W-S,7 chronic gastritis patients single positive for RUT and W-S were eliminated. 60 chronic gastritis patients had successful eradication of Hp,the rate of eradication was 89.55%(60/67),21 cases for chronic superficial gastritis CSG),13 cases for chronic gastritis with atrophy (CAG),15 cases for intestinal metaplasia(IM),11 cases for gastric epithelial dysplasia(DYS).31 patients were followed up the six month. 2. The positive rate of E-cadherin methylation in Hp infected patients was significantly higher than without infections(45% vs. 9.8%,P<0.001). The positive rate of DAPK gene methylation was 50% in Hp infected subjects and 43.9% without infections(P>0.05). 3. The methylated gastric chronic inflammation score of E-cadherin gene was significantly higher than the unmethylated gastric chronic inflammation score(2.16 vs. 1.6 , P=0.017). The methylated gastric chronic inflammation score of DAPK gene was slightly higher than the unmethylated gastric chronic inflammation score(1.96 vs. 1.61,P=0.093). 4. Univariate analysis showed that CpG island methylation of the E-cadherin gene was significantly associated with the presence of Hp infection and the chronic inflammation (P = 0.004 and P = 0.001, respectively). Forward stepwise binary logistic regression analysis showed that no correlation was found between E-cadherin gene methylation and other clinicopathologic parameters,such as age,sex,alcohol,smoking,family history of gastric cancer,but a significant association between E-cadherin gene methylation and Hp infection(OR 7.568,95% CI 2.396-23.906,P=0.001).5. After Hp eradication, E-cadherin gene methylation significantly decreased(45.16% vs. 12.9%,P=0.017). Conversely,DAPK gene methylation did not change significantly after Hp eradication(22.6% vs. 51.6%,P=0.134). 6. Immunohistochemical method displayed that E - cadherin, DAPK protein expressions had gradually downtrend from chronic atrophic gastritis to intestinal metaplasia to dysplasia (P > 0.05). The methylated E-cadherin, DAPK gene existed E- cadherin, DAPK protein expression down regulation or lost expression.Conlusion 1. The positive rate of E-cadherin methylation in Hp infected nonneoplastic gastric mucosal tissues was significantly higher than without infections. There was a significant association between E-cadherin methylation and Hp infection.2. The methylated gastric chronic inflammation score of E-cadherin gene was significantly higher than the unmethylated chronic inflammation score.3. After Hp eradication, E-cadherin gene methylation significantly decreased. Accordingly,our results suggest that Hp may hypermethylate CpG islands and induce tumor suppressor gene expression silencing probably playing a key role in the development of gastric cancer. Eradication of Hp by reversing methylation is expected to become one of the ways to prevent stomach cancer.