Investigate The Regulatory Mechanism Of Adrenomedullin In Gastric Cancer

Objective:Adrenomedullin(AM)is a Japanese scholar first isolated in human resection of pheochromocytoma tissue of a polypeptide,which in normal plasma in a certain concentration exists,widely involved in the expansion of blood vessels and Lung expansion,for the regulation of blood pressure,a variety of hormonal regulation play a role.In recent years,more and more literature reported AM in a variety of tumors in the high expression,and the extension of the tumor have a certain regulatory role.This study validates the expression of AM in gastric cancer and analyzes the relationship between AM expression and clinicopathological features,which is expected to provide a basis for clinical diagnosis of gastric cancer.A series of in vitro experiments have been conducted to explore the effect of AM on the progression of gastric cancer Gastric cancer targeted therapy to provide new basis and ideas.Methods:The expression of AML and its receptor CRLR,RAMP1,RAMP2 and RAMP3 were detected by IHC,and the expression of AMi was detected by immunohistochemistry.The expression of AML1,RAMP1,RAMP2 and RAMP3 were detected by IHC.The expression of AM in BGC cells was observed and the effect of AM on the proliferation,invasion,migration and apoptosis of gastric cancer cells was observed.The signal pathway of AM was explored.The molecular biology technique was used to construct a stable knockdown and overexpression of AM And to observe the effect of autophagy on gastric cancer cell line BGC-823.The specific operation is as follows:1 Collect gastric cancer tissue adjacent to carcinoma tissue and its matching,q RT-PCR analysis AM gene expression level,IHC testing AM positioning.AM in gastric cancer tissue and the expression of tissue adjacent to carcinoma.Using the IHC testing clinical CRLR AM receptors in gastric cancer specimens,express RAMP1,RAMP2,RAMP3.2 Select different differentiated gastric cancer cell lines: BGC-823,MGC,SGC-790,HGC and MKN-45 AM expression level.Build absolute carrier quantitative detection of AM-m RNA expression level.Select AM express the highest amount of plant cells.3 Design and synthesis of specificity in AM small interference RNA sequences,Lipofectamine2000 transfection cells,at the same time set the negative control group and blank control group.After transfection AM-knock si RNA stable low AM,flow cytometry to detect cell apoptosis and the cell’s ability to attack Transwell method and scratch test cell migration ability,tablet clonal proliferation experiment test cell proliferation.4 Verify the JNK,P-JNK,AKT,P-AKT,PKA,P-PKA and related pathways of protein.Validation of BCL2 and BAX,caspase3,cleaved-caspase3 apoptosis related pathways of protein.5 Build GFP-LC3A/B carrier,Lipofectamine2000 transfection cells,at the same time respectively transfection specificity in AM small interference RNA sequences and pc DNA-AM expression vector,set the negative control group and blank control group.Western blot analysis that autophagy related proteins and Beclin LC3A/B.Confocal microscope autophagosome formation.Results:Relative to the tissue adjacent to carcinoma,gastric cancer specimens in AM a marked increase in the amount of protein expression and gene expression.Expression quantity rise in clinical patients survival rate was significantly lower than the amount of patients(P = 0.0258).The expression levels of AM and their receptors CRLR,RAMP1,RAMP2 and RAMP3 in gastric cancer were significantly higher than those in adjacent tissues,respectively,9 score,8 score,6 score,8 score and 8 score,according to the immunohistochemical evaluation criteria.After transfection AM-knock si RNA stable low AM,flow cytometry detection BGC-823 cells apoptosis rate showed a trend of decrease,scratch test BGC-823 cell migration ability,tablet clonal proliferation experiment test cell proliferation capacity reduction.AM after knock stable low,decreased expression of BCL2 and BAX expression quantity increases,caspase3 expression quantity not seen obvious changes,cleaved-caspase3 expression quantity to rise.Verify JNK,P-JNK,AKT,P-AKT,PKA,P-PKA pathways related protein,P-AKT protein decreased obviously.When AM protein is stable after knocking at low and expression,confocal microscope showed autophagosome did not change significantly.Autophagy related proteins,LC3A/B and Beclin did not change significantly.Conclusion:AM and its receptors CRLR,RAMP1,RAMP2,RAMP3 showed a trend of high expression in gastric cancer compared to the nearby tissue adjacent to carcinoma and the proportion accounted for about 65%.Survival analysis also showed that AM expression of negative correlation with survival.AM the expression of quantity and the size of the tumor cells,tumor classification associated with malignant degree of classification。When AM was stable after knocking at low,BGC-823 gastric cancer cell proliferation in vitro culture at a slower pace,cell migration rate lower,increased cell apoptosis,cell aggressivity.This suggests we targeted on low AM express,can inhibit cancer cell proliferation and migration rate,increase the apoptosis rate,and thus of gastric cancer,increase survival time for gastric cancer patients.And signaling pathway,the study found AM exert its influence on cell proliferation,migration apoptosis may be through the largest AKT signaling pathways.AM effect on gastric cancer cell apoptosis may be activated third endoplasmic reticulum apoptotic pathways,under the stimulus of AM endoplasmic reticulum in the stress state,lead to calcium ion balance,activated caspase-12 on one hand,on the other hand promote apoptosis inducing the Bcl-2 family of proteins Bax activation induced apoptosis,ultimately activate downstream apoptotic perform molecular caspase 3 apoptosis.It is a pity that this study did not find that AM effect on the gastric cancer tumor cell autophagy.May be tumor cells themselves the basic level of autophagy is too low,confocal microscope is not detected autophagosome change obviously.