Effect Of CagA Positive H.pylori Infection On The Expression Of MEX3A In Gastric Cancer And Adjacent Intestinal Metaplasia And Its Related Mechanism

ObjectiveThe occurrence and development of gastric cancer is related to Helicobacter pylori（H.pylori）infection,especially the positive cytotoxin associated gene A（cagA）.H.pylori is more closely related to gastric cancer,and its specific pathogenic mechanism is not clear.The muscle excess 3A（MEX3A）of Caenorhabditis elegans is associated with abnormal proliferation and apoptosis of various malignant tumors,and is closely related to protein ubiquitination.It is also involved in Akt,phosphatidylinositol3-kinases（PI3Ks）,mitogen activated protein kinases（MAPKs）and other signal pathways.It is found that the m RNA expression of MEX3A is related to gastric cancer by querying TCGA database,However,its expression in gastric cancer and adjacent intestinal metaplasia,the effect of H.pylori infection on the expression of MEX3A and its related mechanism are not clear.In this study,the expression of MEX3A in gastric cancer and adjacent intestinal metaplasia was detected by immunohistochemical staining;The Effects of H.pylori infection and cagA positive H.pylori infection on the expression of MEX3A in gastric cancer and adjacent intestinal metaplasia;The effects of cagA containing H.pylori on the expression of MEX3A,phosphorylation retinoblastoma（P-Rb）and cell cycle inhibitor protein 21（P21）in gastric cancer cells（HGC-27 and AGS）were detected by co culture,and the related mechanism of cagA positive H.pylori inducing gastric cancer through MEX3A was explored.MethodsIn this study,410 cases of paraffin embedded gastric mucosal biopsy tissues from the Central Hospital Affiliated to Shenyang Medical College,the Second Affiliated Hospital of Shenyang Medical College and the pathology department of China Medical University from 2011 to 2014 were used.After he staining and double-blind pathological diagnosis by two pathologists in the early stage of the research group,182cases of gastric cancer tissue,149 cases of intestinal metaplasia adjacent to cancer and79 cases of superficial gastritis adjacent to cancer were used as the control group.Type I,type II and type III intestinal metaplasia have been identified by mucin staining.H.pylori was identified by PCR and histological methylene blue staining,and cagA subtype was detected by nested PCR.The expression of MEX3A in the tissues of the experimental group and the control group was detected by immunohistochemical method.The expression of MEX3A in the tissues of the experimental group and the control group was detected by immunohistochemical method.H.pylori ATCC:43504（cagA positive H.pylori as the experimental group）was purchased at ATCC and co cultured with H.pylori ATCC:51932（cagA negative H.pylori as the control group）and gastric cancer cells.The half inhibitory concentration of H.pylori co cultured with gastric cancer cells for 24 hours was detected by CCK-8 to determine the optimal number of infections.The expression of MEX3A,P-Rb proteins before and after 0,4,8,12 and24 hours of culture were detected by Western blot to determine the effect on the proliferation of gastric cancer cells.The immunohistochemical results were analyzed byχ~2or Fisher exact test with statistical software IBM SPSS 24.0.The difference was statistically significant（P<0.05）,and the difference was statistically significant（P<0.01）.The relationship between H.pylori cagA genotype and MEX3A expression was analyzed by logistic regression.At least three independent repeated experiments were carried out in cytological experiments.The values were analyzed by Mean±SD,the differences of protein expression levels were analyzed by t-test,and Graphpad prism 7.0 mapping.Results1.Expression of mex3a in different gastric disease groups and multivariate analysis of clinical dataThe positive rate of MEX3A in the control group（21/79,26.58%）was lower than that in the adjacent intestinal metaplasia group（78/149,52.35%）and gastric cancer group（117/182,64.29%）.The difference was statistically significant（p<0.05）.The positive rate of intestinal metaplasia group was significantly lower than that of gastric cancer group（p<0.05）.The positive rate of type I intestinal metaplasia group（3/13,23.08%）was lower than that of type II intestinal metaplasia group（23/61,37.70%）.The positive rates of type I intestinal metaplasia group and type II intestinal metaplasia group were lower than those of typeⅢintestinal metaplasia group（52/75,69.33%）.There was significant difference between the two groups（p<0.05）.There were 141 male patients and 41 female patients in 181 gastric cancer samples.There was no significant difference in the expression of MEX3A between different gender groups（p>0.05）;Because the age distribution of this group was skewed,the median age of 60 years was taken as the boundary,97 cases were older than 60 years and 85 cases were younger than or equal to 60 years.There was no significant difference in the expression of MEX3A between the two groups（p>0.05）.The tumor stage,tumor diameter,degree of differentiation and depth of invasion were grouped according to the relevant standards of Chinese tumor diagnosis^[30]and the p TNM staging standard of gastric cancer in the eighth edition of AJCC.MEX3A in low differentiation group was significantly higher than that in medium and high differentiation group（p<0.01）.After age and sex adjustment,or=2.819,95%CI=（1.083～7.434）;In the group with lymph node metastasis,it was significantly higher than that in the group without lymph node metastasis（p<0.01）.After adjusting for age and sex,or=4.461,95%CI=（2.228～8.931）;It was significantly higher in diffuse gastric cancer group than in intestinal gastric cancer group（p<0.05）.After age and sex adjustment,or=2.620,95%CI=（1.368～5.016）.2.Effect of H.pylori infection,especially cagA positive H.pylori infection,on the expression of MEX3A in gastric cancer and adjacent intestinal metaplasiaThe positive rate of MEX3A in the control H.pylori（+）group（14/79,17.72%）was higher than that in the corresponding H.pylori（-）group（7/79,8.86%）;The positive rate of MEX3A in intestinal metaplasia H.pylori（+）group（56/149,37.58%）was significantly higher than that in the corresponding H.pylori（-）group（22/149,14.77%）（p<0.01）;The positive rate of MEX3A in H.pylori（+）group（88/182,48.35%）was significantly higher than that in H.pylori（-）group（29/182,15.93%）.The difference was statistically significant（p<0.01）.In the control group and intestinal metaplasia group,the positive rate of cagA（+）MEX3A（13/79,92.86%;36/149,64.29%）was higher than that of cagA（-）（1/79,7.14%;20/149,35.71%）;In gastric cancer,the positive rate of cagA（+）MEX3A（65/149,73.86%）was higher than that of cagA（-）（23/149,26.14%）.The difference was statistically significant（p<0.05）.3.IC50 calculated by CCK-8H.pylori strain containing cag pathogenic island was co cultured with HGC-27 and AGS.After the OD value was detected and calculated by enzyme labeling instrument,it was found that the best co culture concentration was the concentration of multiplicity of infection（MOI）=1:100.4.Western BlotIn HGC-27 cells,the expression of MEX3A protein in cagA（+）H.pylori group was significantly higher than that in cagA（-）H.pylori group at 4,12 and 24 hours of co culture（p<0.05）.The expression of p21 protein in cagA（+）H.pylori group was higher than that in cagA（-）H.pylori group at 24 hours（p<0.05）.The expression of p-rb protein in cagA（+）H.pylori group was higher than that in cagA（-）H.pylori group at 4hours（p<0.05）.In AGS cells,the expression of MEX3A protein in cagA（+）H.pylori group was significantly higher than that in cagA（-）H.pylori group at 4,8,12 and 24 hours of co culture（p<0.05）.The expression of p21 protein in cagA（+）H.pylori group was higher than that in cagA（-）H.pylori group at 8 hours（p<0.05）.The expression of p-rb protein in cagA（+）H.pylori group was higher than that in cagA（-）H.pylori group at 4 hours（p<0.05）.Conclusions1.High expression of MEX3A in gastric cancer and adjacent intestinal metaplasia can increase the risk of gastric cancer,and is closely related to poorly differentiated gastric cancer,lymph node metastasis and gastric type gastric cancer;The high expression of MEX3A is closely related to adjacent intestinal metaplasia,especially type III intestinal metaplasia.2.H.pylori infection,especially cagA containing pathogenic Island H.pylori infection,can cause intestinal metaplasia and high expression of MEX3A in gastric cancer,which is a risk factor for intestinal metaplasia and gastric cancer.3.H.pylori infection of cagA containing pathogenic Island caused the increase of MEX3A expression,P-Rb expression at 4 hours and P21 expression at 8 and 24 hours,which was related to the occurrence of gastric cancer.