| Objective: Gastric cancer is the one of the most common malignant tumors,it remains the fifth most common malignancy and the third leading cause of cancer-related death worldwide.The five-year survival rates of gastric cancer patients only range from 20% to 40%.Although the implication of surgical process,various kinds of drugs and radiotherapy in the treatment of gastric cancer truly improved the survival rate,and decreased the recurrence or metastasis risk for gastric cancer.The treatment effect of these multiple methods is still unsatisfactory.Exploring the key molecules and related mechanism could provide novel gastric cancer prevention and treatment direction as well as improve the treatment effect of gastric cancer in some extent. G protein-coupled receptors(GPCR)are the one of the largest membrane protein family and located in the cell membrane.GPCR could be interacted with G protein and transfer multiple extracellular signals and stimulations into cell,which could be involved in a series of pysiological activity,such as the cell proliferation,differentiation,metabolism,migration,secret,cell growth and development,inflammation as well as the immune reaction.Recent studies also indicated that many GPCR family members have been involved in the various kinds of disease,such as the cardiovascular and cerebrovascular diseases,nerves system disease,allergy,tumor and inflammation reaction.Meanwhile,many GPCR family members are also associated with the malignant tumor biological behavior including proliferation,invasion and migration. Neuropeptide S receptor 1(NPSR1),one of the GPCR family member,which was discovered in recent years.Since the neuropeptide S is the endogenous ligand peptide,it is finally named as NPSR1.NPSR1 is widely expressed in human body and many tissues could detect the expression of NPSR1,such as the brain tissue,thymus,lung tissue,bronchi,gastrointestinal tract tissue(esophagus,stomach,small intestine,colon)and peripheral blood mononuclear macrophages and eosinophils.Thus,previous studies reported that NPSR1 may regulate physiological activities such as the neuropsychiatric activities and immune reaction. Currently,few studies have reported the function of NPSR1 in tumor.Previous studies investigated the expression of NPSR1 in the neuroendocrine tumors,and the results showed that the widely expression of NPSR1 could be selected as the potential diagnosis biomarker for the neuroendocrine tumors.However,the mechanism of NPSR1 in neuroendocrine tumors and the expression of NPSR1 in other solid malignant tumor were still unclear.Additionally,previous study found that NPSR1 could modulate the cell proliferation under the stimulation of the ligand peptide NPS.This may provide some evidence for the function of NPSR1 in malignant tumors.Bioinformatics analyses illustrated that NPSR1 showed significant differential expression in gastric cancer sample and some association with clinicopathological characteristics.Based on above considerations,our studies aim to identify the expression of NPSR1 in gastric cancer tissue and further explore the influence of NPSR1 in biological behavior as well as the potential mechanism.Methods: The gastric cancer expression matrix from The Cancer Genome Atlas(TCGA)were used to compared the differential expression of NPSR1 between gastric cancer tissue and normal tissues;and further explore the association with clinicopathological information.256 gastric cancer formalin-fixed paraffin-embedded tissue samples were used to perform the immunohistochemistry(IHC).The results of IHC were used to compare the differential expression of NPSR1 between cancer tissue with adjacent normal tissue and further explore the association with prognosis and clinicopathological information. Western blot analyses were used to evaluate the expression of NPSR1 in gastric cancer cell line AGS,SGC-7901,MGC-803 and gastric cell line,GES-1.To further explore the function of NPSR1,RNAi-mediated knockdown via lentiviral vector was used to decreased the NPSR1 expression in two gastric cancer cell lines,SGC-7901 and MGC-803.The negative control cell lines were also constructed.Western blot and quantitative real time PCR were used to evaluated the transfection efficiency.We compared the cell migration and invasion capabilities between the NPSR1 knockdown cell and negative control cell by using the Transwell chambers experiment.The comparison of cell proliferation capability was evaluated by the Cell Counting Kit-8 experiment and colony formation assay.Flow cytometry analysis was performed to evaluate the cell cycle distribution and the proportion of apoptosis cell. To further explore the potential mechanism and the signal pathway of NPSR1.Western blot assay was used to evaluate the MAPK/ERK signal pathway and their down-stream target gene after knockdown the expression of NPSR1. Variables comparison were used the t-test,rank-sum test and Chi-square test.Kaplan-Meier method was used to evaluate the patient survival and log-rank test was used to compare the survival between different groups.Cox proportional hazards regression analysis was used to identify the independent risk factors.A two-tailed P value < 0.05 was considered as statistically significant in all analyses.All the statistical analyses and image presentation were performed using the SPSS version 21.0,R software(Version:3.5.1)and GraphPad Prism 6.0.Results: The results of TCGA gastric cancer database showed that the NPSR1 mRNA expression of cancer sample was extremely higher than the normal sample,and this significant differential were observed both in paired sample and unpaired samples(P<0.001).Further compared the NPSR1 expression between different clinicopathological showed that the NPSR1 presented significant association with the depth of tumor invasion,TNM stage and tumor grade(P<0.05).Immunohistochemistry assay showed that NPSR1 was higher expression in the cancer tissue,which presented as the higher positive rate in cancer tissue than in the adjacent normal tissue(P<0.001).Meanwhile,higher expression of NPSR1 was positively correlated with advanced T stage,N stage and TNM stages.The survival curves showed that patients with higher NPSR1 expression had worse survival than patients with lower NPSR1 expression,further analyses indicated that the expression of NPSR1 was the independent prognosis risk factor.In the cancer cell line assay,we constructed two cancer cell lines(MGC-803 and SGC-7901)which decreased the expression of NPSR1 steadily.The western blot and quantitative real time PCR validated the transfection efficacy and the results showed the NPSR1 expression in these two cell lines were markedly decreased. The transwell chambers experiment evaluated the capability of the cell invasion and migration.The results showed that less cells transfer into the lower chamber in cells with NPSR1 knockdown when compared with negative control cell.These results indicated that NPSR1 knockdown significantly inhibited the invasion and migration ability of gastric cancer cells.Cell Counting Kit-8 experiment and colony formation assay showed that compared with the negative control cells,the cell growth and the number of colony formation of cells with NPSR1 knockdown were significantly decreased.These results indicated that knockdown NPSR1 could inhibit proliferation ability of gastric cancer cell. Flow cytometry analyses used to evaluate the cell cycle.The results showed that compared with negative control cells,knockdown NPSR1 could significantly increase the cell proportion in G0/1 phase and further decrease the cell proportion in G2/M phase in these two cell lines.These results indicated that the cell cycle may arrest in the G1 phase.Comparing the apoptosis cell proportion and the results showed no significant difference between NPSR1 knockdown cells and negative controls.These results indicated that the knockdown NPSR1 did not induce cell apoptosis. Further western blot analyses evaluated whether knockdown NPSR1 is associated with the MAPK pathway and we examined the expression and phosphorylation of the MAPK pathway signaling molecules ERK(1/2).The results showed that the expression of ERK(1/2)did not show significant change,however,the activation of phosphorylated ERK(1/2)decreased.Additionally,the mRNA and protein expression of Cyclin D1 and CDK4 were also downregulated after the NPSR1 knockdown.These results may indicate that NPSR1 could regulate the MPAK/ERK pathway in vitro.Conclusion: The NPSR1 expression in the gastric cancer tissue was significantly higher than the adjacent normal tissue and associated with the clinicopathological characteristics.Patients with higher NPSR1 expression had worse survival than those with lower NPSR1 expression and higher NPSR1 expression was also the independent prognostic factors.NPSR1 Knockdown could significantly inhibit the cell proliferation,cell invasion and migration,it could also influence the cell cycle.Further analyses indicated that NPSR1 knockdown could regulate the MPAK/ERK pathway in vitro. |
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