Vagus Nerve And M3 Regulate The Development Of Gastric Cancer

Backgroud&objective Gastric cancer(GC)is one of the most common malignant tumors with about 1,000,000 new cases each year,accounting for 10%of new cases of cancer worldwhile.In china,there are approximately 400,000 new cases of it,occupying 42%of the total new cases in the world.The combined therapy based on radical surgery is the main treatment of GC.Despite significant achievements in the technique of gastric surgery,chemoradiotherapy and molecular targeted therapy,the prognosis of GC remains poor,with the five-year survival rate only 20-25%.The main influencing factors of GC including dietary factors,Helicobacter pylori infection,environmental exposures,oncogenes activation,tumor suppressor genes inactivation,tumor microenvironment and immune escape of tumor cell and so on.However,the detailed molecular mechanisms underlying the development of GC are still under investigation.Further analysis of the molecular mechanism underlying it is helpful to identify novel therapeutic targets for better treating GC.Muscarinic receptor M3,a member of G-protein coupled receptors(GPCRs),seven transmembrane,is an acetylcholine(ACh)receptor that regulates the activity of numerous fundamental central and peripheral nervous system functions.Recent studies have identified the activation of the M3 receptor in several cancers and the role in the development of GC.As a member of tumor microenvironment,nerve dominating tumor organ plays an important role in the genesis and development of tumor.Vagus nerve dominates in the innervation of stomach,and it is mainly distributed in the lesser curvature.About 80%of GC occurs in the lesser curvature.We also know that M3 and its ligand ACh are important elements of vagus nerve signal transmission,and M3 receptor is expressed in gastric epithelial cells and glandular cells.So,do GC cells express M3 receptor?Does the M3 receptor regulate the biological behavior of GC?Whether vagus nerve control the development of GC?Does the M3 receptor mediate the effect of vagus nerve on the development of GC?To solve the above problems,this study was performed.Methods 1.The mRNA and protein levels of M3 in 106 human GC tissues and their paired non-cancerous tissues were determined by qRT-PCR and immunohistochemistry.The correlation between M3 expression and clinicopathological characteristics was further analyzed.The co-expression of M3 and choline acetyltransferase(ChAT)were detected by immunohistochemistry and double immunofluorescence confocal microscopy.ACh concentrations in the supernatants of GC cells were measured by high-performance liquid chromatography.2.M3 receptor was activated by exogenous ACh stimulation and the expression of M3 was downregulated by a siRNA.Further functional assays including CCK8 assay,plate clone assay and in vivo tumorigenesis assay were performed.The possible underlying mechanisms were analyzed by flow cytometry,western blot and immunohistochemistry.3.To decrease the activity of vagus nerve,the left trunk of vagus nerve was cut off.The effect of downregualtion of vagus nerve activity on tumorigenesis of GC cells in vivo was determined by an orthotopic models of human GC in nude mice.Further mechanical analysis was performed by whole mouse genome microarray,qRT-PCR,immunohistochemistry and M3 antagonist assay in vivo.Results 1.The M3 receptor was overexpressed in human GC tissues and was correlated with the cancer stage and with lymph node metastasis.We also detected the existence of ACh in the supernatants of GC cells and observed the co-expression of the M3 receptor and ChAT in human GC tissues and cells.2.In vitro,the M3 receptor enhanced the proliferation induced by acetylcholine in human GC cells,whereas the knockdown of M3 inhibited cell proliferation.Furthermore,M3 knockdown caused G2/M phase cell cycle arrest and induced apoptosis in human GC cells.In vivo,M3 knockdown suppressed tumorigenesis and promoted apoptosis in GC xenografts.3.Downregualtion of vagus nerve activity inhibited human GC cells tumorigenesis in nude mice and decreased M3 expression in mice gastric tissues.Moreover,M3 antagonist suppressed the development of gastric cancer in nude mice.Conclusion 1.Cholinergic signaling pathway is present in GC.M3 expression level is increased in GC and correlates with the development of GC.2.M3 promotes GC cell proliferation in vitro and enhances tumorigenesis of GC cells in vivo,the underlying mechanism is possibaly by MAPK pathway,cell cycle regulation and apoptosis pathway.3.Downregualtion of vagus nerve activity suppresses the development of GC in nude mice.M3 probably mediates the effect of vagus nerve on development of GC in nude mice.