Roles And Mechanisms Of GnRHR In GC Development, Differentiation,invasion And Metastasis

Objective: Correlation of GnRHR in gastric cancer tissues with the prognosis of gastric cancer patients was analyzed by detecting GnRHR expression in gastric cancer tissues. To explore the roles and molecular mechanisms of GnRHR in gastric cancer cell development, differentiation, invasion and metastasis.Methods: GnRHR mRNA was examined in tumor and matched non-tumor tissues from 48 gastric cancer patients by Real-time PCR. The GnRHR protein expression was performed by immunohistochemical. The relationship between the GnRHR expression and GC TNM, lymph node metastasis or distant metastasis was analyzed by chi-square. Kaplan Meier and survival analysis was used to analyze the rank test between GnRHR expression and the prognosis of gastric cancer patients. According to tumor differentiated degree, tissue specimens of 48 gastric cancer patients were divided into two groups(poor differentiation and well differentiation), and expressions of GnRHR and EGFR were semi-quantitatively analyzed through SP immunohistochemical method and the method of in situ quantification. We observed GnRHR expression in three different differentiated gastric cancer cells by Western blot. MTT was used to detect gastric cancer cells proliferation after cells were treated with different concentrations of triptorelin acetate(1×10-10,1×10-9,1×10-8,1×10-7,1×10-6mol/L). Expression of GnRHR and differentiation related proteins were detected by Western blot. Transwell Chambers was used to observe invasive and migrational ability of gastric cancer cells after GnRHR was activated by triptorelin acetate.Western blot was used to detect the expressions of EMT associated protein E-cad,vimentin and snail after GnRHR activated. Phosphorylation of EGFR was induced by protein tyrosine phosphatase inhibitor Sodium orthovanadate and then MTT was used to detect cell proliferation,Western blot was used to detect proteins of proliferition, differentiation as well as the EMT related proteins.Results:we respectively detected GnRHR mRNA and protein expression of gastric tumor tissues and matched non-tumor tissuesfrom 48 patients by RT-PCR and immunohistochemicalion for studying the role of GnRHR in gastric cancer. We found that the expression of GnRHR mRNA was higher in gastric tumor tissues than matched non-tumor tissues(-10.06 ± 1.28 vs-12.43 ± 1.33, p<0.05). GnRHR protein was mainly located in cytoplasm of gastric cancer cells. The staining of GnRHR was not detected in tumor tissues in more than half of 48 gastric cancer patients(52.08%, 25/48). To explore the correlation between GnRHR and clinical parameters as well as prognosis of gastric cancer patients. We collected the clinical and follow-up datas of 48 gastric cancer patients. By Statistical software, we found that GnRHR mRNA expression was associated with lymph node metastasis, distant metastasis, and TNM stage. GnRHR mRNA expression was associated with gastric cancer patients’ overall survival. Besides, low expression of GnRHR mRNA(-ΔCt <-10.06) associated with gastric cancer patients’ poor overall survival(p = 0.003; HR = 0.42, 95% CI: 0.22-0.87). Tumor tissue samples of 48 gastric cancer patients were divided into good and poor differentiation groups according to the degree of tumor differentiation.GnRHR and EGFR positive immune response are different in different differentiation carcinoma tissues. GnRHR positive expression is 23 cases(47.92%) and EGFR positive expression is 26 cases(54.16%). By In situ quantitative, we found that GnRHR positive expression is 20 cases good and 3 cases poor differentiation,and with tissue differentiation degree higher and higher. However EGFR positive expression is 13 cases good and 13 cases poor differentiation,and with tissue differentiation degree higher and lower(p<0.05). In order to further investigate the relationship between GnRHR and EGFR, and the molecular mechanism about GnRHR roles in gastric cancer cells development, differentiation, invasion and metastasis.We studied the GC cells proliferation, differentiation, invasion and metastasis from cell levels. The results showed that different concentrations and different time of triptorelin acetate can inhibit gastric cancer cells proliferation.Western blot showed that GnRHR inhibits gastric cancer cells proliferation through the EGFR- ERK pathway.In addition, the results showed that GnRHR expressed in different differentiation degree of gastric cancer cells is different, with gastric cancer cells differentiation higher and GnRHR expression higher.Western blot further showed that GnRHR regulates the gastric cancer cells differentiation through EGFR- CDK1 pathways. GnRHR expression in gastric cancer tissues is associated with tumor metastasis.To explore GnRHR effects on gastric cancer cells invasion and metastasis through cellular levels,After triptorelin acetate Inducing GnRHR expression in gastric cancer cells, we detected ability in gastric cancer cells migration and invasion by transwell little room.The results showed that the migratory and invasive ability of cells decreases after triptorelin acetate treatment.It implied that inducing expression of GnRHR can inhibit gastric cancer cells migration and invasion. western blot showed that GnRHR regulaed down level of EGFR phosphorylation and further inhibits expression of vimentin and snail protein.The result implied that GnRHR regulates gastric cancer cells invasion and metastasis through EGFR-EMT pathways.Conclusions:GnRHR expression in gastric cancer tissues is related with tumor metastasis, TNM stage and prognosis in gatric cancer patients. GnRHR and EGFR expression in related to the differentiation of gastric cancer tissue. GnRHR play a role in the gastric cancer development, differentiation, invasion and metastasis through regulating EGFR.