The Role And Mechanism Of NTSR1 In Gastric Cancer Proliferation And Metastasis

There is a high incidence of gastric cancer(GC)in China,and the morbidity and mortality is in the top four in malignant tumors.Nearly half of patients at the time of diagnosis had been at an advanced stage,thus lost the chance of surgical treatment.Prognosis of GC is still dependent on the traditional pathology index.Therefore,it is imperative to study the related molecular mechanism in the development and progression of GC,which involves a series of oncogene activation and/or inactivation of tumor suppressor genes and is a multi-stage and multi-dimensional process.Screening of new oncogenes and studying their mechanisms of action have important clinical value in the early diagnosis of GC biomarkers and potential molecular therapeutic targets.Neurotensin(NT)is a neuroendocrine peptide widely distributed in the human body.NTSR1(neurotensin receptors 1,NTSR1)is a highly specific receptor of NT.Recent studies have shown that NT/NTSR1 is highly expressed in various tumor tissues,such as pancreatic cancer,prostate cancer,breast cancer,colon cancer,lung cancer and head and neck cancer.NT/NTSR1-mediated signal transduction pathways play an important role in tumorigenesis and progression.It can promote tumorigenesis,proliferation,migration,invasion and survival.NT/NTSR1-mediated multiple signaling pathways have a significant role in promoting the development of tumors.Recent studies have shown that NTS/NTSR1 signaling can induce EGFR(epidermal growth factor receptor)activation in colon cancer,lung cancer,and prostate cancer,thereby promoting cancer cell division,proliferation and growth.HER2(human epidermalgrowth factor receptor-2)is one of the important members of EGFR family.HER2 gene amplification plays an important role in the development and progression of GC.When HER2 and EGFR are coexpressed,the former often binds to the ligand-activated latter to form a dimmer and the heterodimer tends to have higher re-utilization,stability and ability to conduct signals compared to EGFR homodimers.At present,there is little literature on NTSR1 gene and EGFR/HER2 correlation study.A study has found that sustained stimulation of NTSR1 can leads to EGFR and HER2 overexpression and activation in lung cancer.In summary,NTSR1 gene may play an important role in the development and progression of gastric cancer.At present,there is no research on the clinicopathological significance of NTSR1 expression and on its mechanism of development in gastric cancer.The purpose of this study is to explore the significance of NTSR1 expression in gastric cancer,try to reveal its role in the proliferation of gastric cancer,to explore the role of Cancer Phenotype signaling pathway and EGFR/HER2 signaling pathway in it,and to further provide a theoretical basis for study on the mechanism of NTSR1 in the development and progression of gastric cancer.ObjectiveTo detect the expression of NTSR1 in GC tissues and adjacent tissues and to explore its relationship with clinicopathological features and prognosis.The expression of NTSR1 in gastric cancer cell lines was detected.The RNAi technique was used to silence the expression of NTSR1 gene in GC cell lines and to investigate the changes of proliferation and invasion of gastric cancer cells after interfering expression and to explore the mechanism of NTSR1 on the proliferation,apoptosis and invasion of GC cells initially.MethodsThe expression of NTSR1,EGFR and HER2 in 233 gastric cancer tissues and adjacent tissues were detected by immunohistochemistry Envision two-step method.To analyze the relationship between the expression of NTSR1 protein and the clinicopathological parameters of patients with gastric cancer and the prognosis of 210 patients and its relationship with EGFR and HER2.The expression of NTSR1 mRNA in SGC-7901,MKN-45,MKN-74,AGS and MGC-803 gastric cancer cell lines was detected and the cell lines with high abundance were selected as the experimental subjects.Three NTSR1 shRNA expression vectors were transfected into MKN-45 cells by RNAi(Lentivirus infection).Real-time PCR was used to detect the inhibitory effect of shRNA on NTSR1,and the shRNAs with the highest interference efficiency were used to construct stable strains.MTT assay was used to observe the proliferation of gastric cancer cells after NTSR1 gene silencing.The effect of silencing NTSR1 gene expression on gastric cancer cell apoptosis was detected by flow cytometry.The effect of NTSR1 gene expression on invasive ability of gastric cancer cells was observed by cell invasion assay(Corning Invasion Kit).To detect the expression of related genes in the cancer Phenotype signaling pathway and western blot was used to detect the effect of NTSR1 on EGFR / HER2 downstream signaling factors pErk,p-p38 and pAkt and to explore the mechanism of NTSR1 on the proliferation and invasion of gastric cancer cell lines.Results1.Immunohistochemical analysis showed that 161 cases(69.1%)were positive in GC,but only 31 cases(13.3%)in normal gastric tissue,the others were all negative,and the expression was significantly up-regulated(P<0.01).2.NTSR1 overexpression was positively correlated with histological grade(P= 0.048),T stage(P = 0.001),TNM stage(P= 0.013)and lymph node metastasis(P= 0.000).It was not associated with gender,age,tumor size and Lauren's classification.3.There was significant correlation between the expression of NTSR1 and EGFR and HER2(P=0.045 and 0.002)and a significant correlation between the expression of EGFR and HER2(P = 0.003)in 233 gastric cancer patients.4.The 1-year and 3-year survival rates of 210 patients with gastric cancer were 78.9% and 68.0%,respectively.By Log-rank test,High expression of NTSR1(P=0.000),low or differentiated carcinoma(P=0.014),diffuse Lauren's classification(P=0.004),high T stage(P=0.000),high expression of NTSR1 P=0.026),lymph node metastasis(P=0.000)and high clinical stage(P=0.000)had a poor prognosis.5.The expression abundance of NTSR1 was high in MKN-45,moderate in SGC-7901 and low in MKN-74,AGS and MGC-803.6.NTSR1 shRNA expression vector was successfully constructed and the stable cell lines were constructed.The proliferation of MKN-45 cells was significantly down-regulated by NTSR1 shRNA(P<0.01).7.The apoptosis rate of MKN-45 was significantly increased by NTSR1 shRNA(P<0.05).8.The expression of NTSR1 and the invasion and metastasis of MKN-45 were significantly decreased by shRNA(P<0.05).9.In MKN-45 cells,the expression level of p27 Kip1 protein in cancer Phenotype signaling pathway was significantly up-regulated after NTSR1 shRNA was transfected into MKN-45 cells,at the same time,EpCAM,HER2 / ErbB2,EGF Receptor and Met also showed a significant downward trend.10.The expression of pErk,p-p38 and pAkt in EGFR/HER2 signaling pathway was significantly down-regulated by shRNA-induced NTSR1 expression in MKN-45 cells(P<0.05).ConclusionThis study shows that NTSR1 plays an important role in the occurrence,invasion and metastasis of gastric cancer,NTSR1 can promote the proliferation and invasion of gastric cancer cells.NTSR1 may promote the proliferation,invasion and metastasis of gastric cancer cells by regulating the EGFR / HER2 signaling pathway and P27 Kip1,EpCAM,Met and other Cancer Phenotype signaling pathways.