The Regulation And Significance Of NHE1 In Acidic Micro-environment Of Gastric Carcinoma

Background and Objective:Gastric carcinoma is one of the most common malignant tumors of digestive system in China, it's mortality rate ranks first. Peritoneal metastasis of gastric cancer is the most important death cause in patients.Gastric cancer cells and peritoneal microenvironment interaction results peritoneal metastasis.Studies have shown that intraperitoneal pH is acidic,tumor cells inner pH is alkaline.NHE1 (Na⁺-H⁺ exchanger1) plays an important role in regulating the up system.NHE1 is one of the important mechanisms of regulating intracellular pH. Intracellular pH Stability is a prerequisite for maintaining normal function of cells. NHE expression is significantly increased in tumor cells, it is closely related to the tumorigenesis and metastasis. NHE has 9 subtypes, named NHE1 9. NHE gene is the key regulation of intracellular acid-base balance, it may plays an important role in the metatasis of gastric carcinoma.Scientists has found that NHE1 protein C-terminal membrane area containing a large number of lysine and arginine sequence, which could Interact gene Vil2 directly, gene Vil2 are encoding Protein Ezrin.Ezrin is a member of the ERM family,It is involved in mediating cell - cell / cell - extracellular matrix adhesion complex formation. Some studys showed that tumor cells with Ezrin protein large expression reduce the surface adhesion complex , there by promoting metastasis. Some studys have the opposite view.NHE1 and Ezrin expression in Hepatic carcinoma, Esophageal cancer, Breast cancer and other tumors have been already reported in the literature related, but their expression and correlation in gastric carcinoma are rare reported. NHE1 regulates the intracellular pH to the dynamic balance to influence cell morphology and function, to participate in many complex physiological and pathological processes. Ezrin expression or abnormal activation regulated pathway affect cell function in many ways, change to the malignant phenotype, increased invasive capacity. The research of NHE1, Ezrin protein expression in gastric cancer, is significant in promoting combating tumor diseases, inhibit cell malignant transformation and reducing tumor metastasis. Laparoscopic technology as the representative of minimally invasive surgical technology is the mainstream in surgery , whether CO₂ pneumoperitoneum promote gastric cancer peritoneal metastasis or not is the focus attention problems. Do the research of NHE1, Ezrin mRNA expression in human gastric cancer MKN45 cells at different CO₂ pneumoperitoneum pressure and different time points in cultured , investivate CO₂ pneumoperitoneum will or will not promote gastric cancer peritoneal metastasis, will provide a theoretical basis of clinical endoscopic treatment of gastrointestinal cancer. This study is to select a full range of clinical and pathological data of 60 cases of gastric cancer specimens, corresponding to 60 cases of normal gastric mucosa as control group, NHE1 and Ezrin mRNA expression were detected by RT-PCR, NHE1 and Ezrin protein expression were detected by Immunohistochemistry and Western blot. Cultivate human gastric cancer MKN45 cell at different CO₂ pneumoperitoneum pressures, different time points, NHE1 and Ezrin protein expression were detected by western blot, to investivate the regulation and significance of NHE1 in acidic micro-environment of gastric carcinoma.Methods:1. Samples collection60 cases of gastric cancer patients studied admitted in the General Surgery Center, Southwest Hospital, the First Affiliated Hospital, Third Military Medical University, June 2008-May 2009. gastric cancer tissues were resected surgically, at the same time to take the edge away from the tumor≥5cm as the normal gastric mucosa, All specimens were placed in liquid nitrogen tank after the sodium chloride solution rinse. Male 46,female 14;Age 34 -78 years, mean age 58.0 years. All gastric cancer patients before surgery had not received radiotherapy and chemotherapy. With WHO histological classification, Well differentiated adenocarcinoma 18 cases, differentiated adenocarcinoma 28 cases, poorly differentiated adenocarcinoma 14 cases; lymph node metastasis 53 cases, no lymph node metastasis 7 cases. Under the 13th edition of Japan Statute staging of gastric cancer treatment, stage I-II 19 cases, stage III-IV 41 cases.2. Methods. NHE1,Ezrin mRNA and protein in Gastric cancer and normal gastric mucosa were detected by RT-PCR, Immunohistochemistry and Western blot analysis, semi-quantitative analysis of NHE1, Ezrin expression, Compared differences between cancer and normal tissue with theχ² test, analyze correlation between NHE1 and Ezrin with Pearson correlation test, analyze the statistical significance of NHE1 and Ezrin in Age, gender, tumor invasion depth, lymph node metastasis, differentiation stage.3. The human gastric carcinoma MKN-45 cells were divided into experimental and control groups. In the experimental groups, the MKN-45 cells were exposed to CO₂ with different pressures ( 5, 10, or 15 mmHg) for 4 hours; The cells exposed to CO₂ with 15mmHg for 0.5 hour,2 hours,4 hours and cultivated in normal environment for 1 hour after being treated for 4 hours. The cells in the control group was cultivited in normal environment. The expressions of NHE1 and Ezrin of all groups were detected by Western blot.Results:1. RT-PCR results indicated that NHE1 mRNA level was increased in 48 cases of gastric carcinoma and 23 cases of adjacent normal tissue, Ezrin mRNA level was increased in 21 cases of gastric carcinoma and 33 cases of adjacent normal tissue.2. Immunohistochemistry results indicated that NHE1 protein level was increased in 44 cases of gastric carcinoma and 18 cases of adjacent normal tissue, Ezrin protein level was increased in 22 cases of gastric carcinoma and 45 cases of adjacent normal tissue.3. The NHE1 and Ezrin expressions of all gastric tumor MKN45 cell groups exposed to CO₂ under 15mmHg were not significantly different from each other.Conclusion:1. NHE1 mRNA and protein expression were significantly higher in gastric cancer tissue than normal gastric tissues, Ezrin mRNA and protein were significantly lower in gastric cancer tissue than normal gastric tissues, indicate that NHE1 and Ezrin could be used as one of the gastric carcinoma development genes.2. The expressions of NHE1,Ezrin mRNA and protein were related to the malignant progression of gastric carcinoma, lymph node metastasis and staging between groups (P<0. 05), However, no statistical difference was seen in patients age, gender and differentiation between groups (P>0.05), indicate that NHE1 and Ezrin could be used as one of the predictive factors for monitor. 3. The CO2 pneumoperitoneum pressure used under 15 mmHg may have no influence on gastric cancer cell proliferation and metastasis through NHE1 and Ezrin.