The Role Of Regeneration Iα (RegIα) In Gastric Garcinogenesis And The Possible Regulatory Mechanisms

Gastric cancer is a very common disease and remains the second leading cause of cancer mortality in China. According to Correa's hypothesis, atrophic gastritis, intestinal metaplasia (IM) and dysplasia are precancerous lesions with a considerable risk in developing into gastric carcinoma. In fact, there were studies proved that chronic gastric inflammation may precede or lead to gastric cancer. Thus, we preposed a new hypothesis of gastric carcinogenesis as followes:the long-term inflammation of the gastric mucosa can lead to the destruction of committed stem cell in the deep mucosa, and the bone marrow stem cells could colonize at the damaged glands. The change of immunity and inflammation in gastric gland, will lead to incomplete repair of the damaged glands. The cancer-related oncogenes may be activated and produced functional proteins when responsed to local microenvironment, which effect on cell dysplasia and tissue abnormal repair, and eventually carcinogenesis. Hence, make clear of the key regulatory factors from chronic gastrity to gastric cancer and intervene the gastric precancerous lesions are the important research orientation in gastric cancer prevention. Indeed, studies have showed that growth factors such as epidermal growth factor (EGF), transforming growth factor-a (TGF-α), vascular endothelial growth factor (VEGF) and many other growth factors participated in the repaire of gastric mucous imflamatory damage. And over-expression of these growth factors is related with proliferation, metastasis and invasion of gastric cancer cells. Accordingly, further screen new growth factors and studying the related regulatory mechanisms became the "hotspot" research of gastric cancer.The regenerating gene (Reg) is a new growth factor which was isolated in regenerating pancreatic islet cells by Yonemura et al. Reg protein was found to be activated during the pancreatic islets regenerative process, suggesting it was an endogenous growth factor in pancreatic islets. Human RegⅠis a protein containing 166 amino acids including a signal peptide. Then, RegⅠwas found produced in gastric mucous and RegⅠαwas the major member of the Reg family expressed by enterochromaffin-like (ECL) cell in the stomach, which played an role in the repaire of gastric mucous damage. Previous studies showed that RegⅠαwas over expressed in H. pylori induced gastritis and gastric ulcer lesions. And there's also reports showed RegⅠαwas expressed in some gastric cancers. In addition, there were studies showed that RegⅠαpromoted the growth and differentiation of the gastric cells in the neck zone, suggesting it was a potent regulatory agent of progenitor cells of the gastric fundic mucosa, and maybe related with gastric carcinogenesis. Therefore, a systematic study of RegⅠαfrom chronic gastritis, gastric precancerous lesion to gastric cancer has a very important meaning.1 Material and Methods1.1 Detection of the expression of Regl a in clinical samples with different pathological state and gastric cancer cell lines.Gastroendoscopy biopsy sample of with different pathological state were collected and gastric carcinoma sample were form tissue bank of Sir Run Run Show Hospital. SYBR-Green real-time quantitative reverse transcription PCR (Q-PCR) was used to detect the mRNA expression of RegⅠαin tissues sample of normal, chronic active gastritis (CAG). intestinal metaplasia (IM), dysplasia and gastric cancer, as well as in 6 gastric cancer cell lines.1.2 Establish a Regla over-expressed gastric cancer cell line and observe the cell proliferation and apoptosisWe constructed a RegⅠαover-expression plasmid named pIRES2-RegⅠα-EGFP. The plasmid was transfected and selected by G418 in MKN28 cell with pIRES2-EGFP as a negative control. The stable transfected cell activity at 0.24,48,96 hour were estimated by MTT. The stable transfected cells were induced by 0,100,200,800μmol/L H2O2 respectively and cell activity at 2,6 hours were detected by MTT. Flow cytometer (FCM) was used to detect cell apoptosis induced by 200μmol/L H2O2. The protein expression level of Bad, Bcl-xL and caspase3 were detected with western blot.1.3 The expression of Regla and gastrin in active gastritis model in rats.An experimental chronic gastritis rat model was established by administration intermixture of 2% sodium salicylate and 30% alcohol,20mmol/L sodium desoxycholate,0.1% ammonia in stead of water, combining irregular feeding. After 10 weeks, the rats were anaesthetized by 4% chloral hydrate and 3ml blood was drawn from arteria and gastric mucosa was preserved. The inflammation of gastric mucosa was estimated by H&E stain. Serum gastrin level was detected by ELISA, and Regl and RegR mRNA expression of gastric mucosa were estimated by Q-PCR and western blot was used to detect the RegⅠprotein expression.1.4 The expression of Regla andβ-catenin after the stimulation with gastrin in AGS cells.Different concentrations of Gastrin-17 (G17) were treated with AGS cells, and the proliferation activity was detected by MTS and the RegⅠαmRNA was assayed with Q-PCR.10-6 and 10-7 mol/L G17 was stimulated for 24h and 72h respectively, and (3-catenin in with total purified protein, cytoplasm protein and nuclear were detected by western blot.2 Results:2.1 RegⅠαover-expression was involved in gastric carcinogenesis and in gastric cancer and precancerous lesions related with poor differentiation in gastric cancer.The mRNA level of RegⅠαwere significantly elevated in active gastritis (n=20), IM (n=40), dysplasia (n=17) and gastric cancer (n=30) when compared with which in normal gastric tissue (n=25) (0.62±0.45/2.68±0.60/2.11±0.42/2.61±0.71/2.47±0.53; P＜0.01, P＜0.05). Morever, the RegⅠαmRNA level were showed highly expression with patients in gastric cancer phaseⅢandⅣwhen compared with which of phaseⅠandⅡ(3.06±0.24/2.98±0.36/1.04±0.24; P＜0.01). In six gastric cell lines, higher RegⅠαexpression was noted in low-differentiated AGS and MKN45 cancer cells (MKN45/AGS/MKN28/NCI-N87/BGC-823/SGC-7901,3.24/3.77/-12.61/-3.70/-11.86/-5.39).2.2 RegⅠαover-expression enhances gastric cancer cell growth, and which mediates an anti-apoptotic effect in gastric cancer cells via Bad/Bcl-xl/Caspase3 pathway.We successfully established a RegⅠαover-expression cell line in MKN28. The cell growth activity of the RegⅠαover-expression cell were significantly higher than control cells (OD value:0.60±0.061/0.51±0.012, P＜0.05; 0.79±0.041/0.67±0.034, P<0.01). When induced by H2O2, the RegⅠαover-expression cell showed stronger anti-apoptotic ability (OD value:1.73±0.067/1.44±0.131, P＜0.01). The protein express of Bad and caspase3 in RegⅠαover-expression cells were decreased after induction with 200μmol/L H2O2 for 6h, while the expression of Bcl-xL was significant increased.2.3 RegⅠαover-expression was related with hypergastrinemia in active gastritis rats.The chronic gastritis rat model was successfully made and identified by H&E stain. The average serum level of gastrin in active gastritis in rats were significantly elevated when compared with control group(375.0±103.9/209.6±48.2ng/ml:P0.05).however,the RegR was significantly elevated in model rats(0.34±0.31/-0.31±0.22,P<0.05).2.4 Gastrin stimulates RegⅠαexpression, and inducesβ-catenin nuclear translocation in AGS cells.The cell proliferation of AGS was increased when increasing the concentration of G17(P<0.01).RegⅠαmRNA level was increased 4.18 folds when stimulated with 10-6mol/L G17 in AGS cells. We also found that the nuclearβ-catenin protein was elevated with the stimulation with 10-6mol/L G17, on the contrast, the cytoplasmβ-catenin protein were significant dropped,which possibly indicated of theβ-catenin nuclear translocation with gastrin stimulation in AGS cells.3 Conclusion3.1 The expression of RegⅠαwas signinficantly elevated in active gastritis,gastric precancerous lesions and gastric cancer.The high expression of RegⅠαwas adapted to gastric mucosa repairment with the condition of damage. Besides, RegⅠαover-expression was probably involved in gastric carcinogenesis.This highlighted that RegⅠαcould be a new potential growth f.actor in the progression from precancerous lesion to gastric cancer.3.2 The expression of RegⅠαwas related with the clinical stage of gastric cancer and gastric cancer cell differentiation.RegⅠαmediated an anti-apoptotic effect in gastric cancer cells via Bad/Bcl-xl/Caspase3 pathway and premoted cell proliferation.It is indicating that RegⅠαmay be a potential biomarker of gastric cancer. 3.3 RegⅠa expression in stomach was related with hypergastrinemia. Gastrin could promote RegⅠαexpression andβ-catenin nuclear translocation in vitro study. It suggested that regulation effect of gastrin on RegⅠαmay throughβ-catenin pathway.