The Effect Of Atrial Natriuretic Peptide Receptor A On The Proliferation And Death Of Gastric Cancer Cells And Its Mechanism

Aim:Gastric cancer is the fifth common cancer type and the second leading cancer-related death.There are nearly 951,000 newly diagnosed cases and almost 723,000 gastric cancer-related deaths.Among those huge number of new cases,over 70%were diagnosed in developing countries and over 50%were in East Asia(mainly in China).The epidemic of gastric cancer in China is still very serious.Many factors,for example,the gene background,the Helicobacter Pylori infection,smoking,dietary habits etc.could contribute to the development of gastric cancer.Gene regulation is very important in gastric cancer promotion,however,we know little about the mechanism underlying gastric cancer progression.Atrial natriuretic peptide(ANP)is a well-studied natriuretic hormone that regulates body fluid volume and blood pressure.The biological effects of ANP are mainly exerted through activation of the natriuretic peptide receptor A(NPRA).Several studies have indicated that ANP-NPRA signaling can also act as an autocrine and paracrine hormone to regulate cell proliferation,cell viability and tumor metastasis,in different organs.Studies indicate that ectopic expression of NPRA is associated with the malignant behaviors of several cancer types,and knockdown of NPRA induces impaired proliferation.The ligand and receptor is expressed in human gastrointestinal tract,and the nature of NPRA that act as a membrane-bound receptor,NPRA could be implicated in human gastric cancer regulation and be used as a treatment target.Methods:Through the construction of gastric cancer tissue microarrays(TMA)and the immunohistochemical analysis combined with the clinical and pathological data to see the correlation of NPRA and clinical features.According to the results,we employed several in vivo.and in vitro.assessments to see the NPRA effects.Cell counting kit 8(CCK-8),colony formation and flow cytometry were used to assess cell proliferation and viability;The transmission electron microscope(TEM),confocal microscope,western blot analysis were used to assess the autophagy level.Autophagy was regulated through administration of 3-MA and chloroquine(CQ)and transfection of siRNAs targeting proteins important in autophagy.Autophagy regulation was employed to assess the protective or lethal role of autophagy after NPRA inhibition.In vivo.analysis was also employed to assess the function of NPRA in vivo.We next analysis possible signals that transduced by NPRA.JC-1 staining and TEM were used to assess the mitochondrial function.DHE and DCFHDA staining and HPLC,flow cytometry analysis to evaluate the ROS level in gastric cancer cells.ROS scavenger NAC were administrated to see whether ROS is a signal that transduced by NPRA inhibition.Western blot was utilized to determine the signaling pathways in NPRA-dependent gastric cancer regulation.Results:NPRA expression was associated with tumor size and disease stage in gastric cancer,and NPRA inhibition induced robust cell death and G2/M cell cycle arrest in a ROS-JNK-dependent manner.At the same time,ROS-AMPK induced autophagy to protect cancer cells from cell death,and inhibition of autophagy enhanced cell death in a caspase-dependent manner.We identified the possible mechanisms by which NPRA signaling regulates cell proliferation and death,and this could help to control gastric cancer progression by eliminating cancer cells and blocking cytoprotective mechanisms.Conclusion:NPRA inhibition resulted in impaired proliferation and viability of gastric cancer,and has the potential to be the target of cancer treatment.