Expression、functions And Mechanisms Of GPR12 In Hypopharyngeal Carcinoma,laryngeal Carcinoma And Esophageal Carcinoma

BackgroundHead and neck cancer(HNC)is the seventh most common cancer in the world.Its incidence accounts for 3%of all cancers.Among them,head and neck squamous cell carcinoma(HNSCC)is the main pathological type,accounting for about 90%of head and neck cell carcinomas.Among head and neck squamous cell carcinomas,hypopharyngeal squamous cell carcinoma(HSCC),laryngeal squamous cell carcinoma(LSCC)and esophageal squamous cell carcinoma(ESCC)are the main types.HSCC accounts for 3%of head and neck cancers.It is difficult to diagnose and has a very poor prognosis.It is usually found in the middle and late stages,and its 5-year overall survival rate is only about 30-35%.LSCC is the cancer with the highest incidence in head and neck squamous cell carcinoma.It mainly includes supraglottic type,subglottic type and glottal type and the glottal type is the most common type.Due to the uncertainty of the incidence of laryngeal cancer and the unusually complex anatomical characteristics,the early detection of laryngeal cancer is difficult.As one of the most common tumors in the world,esophageal cancer has a high incidence.Among them,esophageal squamous cell carcinoma has the highest incidence.Esophageal cancer has a poor prognosis.In recent years,although people continued to improve surgery and treatment methods,its five-year survival rate was still very low.These three types of cancers are usually poorly differentiated.And esophageal and hypopharyngeal cancers are prone to early lymph node metastasis.Therefore,it is urgent to explore the pathogenesis of hypopharyngeal cancer,laryngeal cancer and esophageal cancer,and seek strategies to improve the prognosis of patients and increase the long-term survival rate.G protein coupled receptor 12(Gpr12)is an orphan receptor of the A family of G protein coupled receptors(GPCR).GPR12 has 334 amino acids,which has high similarity with GPR3 and GPR6.So they may have the same function and ligand.In the past research,people found that GPR12 is mainly expressed in the central nervous system,and they are related to the occurrence of central nervous system-related diseases.For example,studies have shown that GPR3,GPR6 and GPR12 are closely related to the onset of Alzheimer’s disease and Parkinson’s syndrome.Besides,it has been reported that GPR3 and GPR12 has the function of blocking meiosis.There have been a large number of literature reports on the pathogenesis of GPR12 in neurological diseases,but its function and mechanism in HNSCC are still unclear.Therefore,in the present study,we aim to explore the expression of GPR12 in hypopharyngeal cancer,laryngeal cancer and esophageal cancer tissues compared with their corresponding normal adjacent tissues,and to explore the effect of GPR12 on hypopharyngeal cancer,laryngeal cancer and esophageal cancer cells’migration,proliferation,cells cycle,apoptosis,etc.,and its possible mechanism.MethodTCGA and GEO databases were used to investigate the expression of GPR12 in head and neck tumors such as hypopharyngeal carcinoma,laryngeal carcinoma and esophageal carcinoma.QRT-PCR was adopted to verify the expression of GPR12 in three cancer tissues to determine the differential expression of GPR12 in several cancers.Afterwards,cell function experiments were carried out to study the function of GPR12 in three tumor cell lines,wound healing and transwell assays were used to study the function of GPR12 on the migration of three kinds of tumor cells,flow cytometry analysis and Caspase-Glo(?)3/7 assay to study the effect of GPR12 on cell apoptosis.The GSEA database was used to analyze the relationship between GPR12 and tumor-related progression genes,and finally the expression of proteins related to migration and apoptosis was measured by Western blot.Result1,According to the analysis of GPR12 in the TCGA and GEO databases,the expression of GPR12 in different head and neck cancers was studied.We found that the expression of GPR12 is down-regulated in hypopharyngeal cancer,laryngeal cancer,esophageal cancer,oral cancer and other head and neck cancer tissues compared to their adjacent normal tissues.Meanwhile it was decreased in cancer.2,We further carried out real-time quantitative PCR research to study the expression of GPR12 in hypopharyngeal carcinoma,laryngeal carcinoma and esophageal carcinoma compared to their normal mucosal tissue.The result showed that in 18 pairs of hypopharyngeal cancer tissues,the expression of GPR12 in 17 pairs of hypopharyngeal cancer tissues was decreased.Among 12 pairs of laryngeal cancer tissues,the expression of GPR12 in 10 pairs of laryngeal cancer tissues were decreased.Meanwhile,among 11 pairs of esophageal cancer tissues,the expression of GPR12 in 10 pairs of esophageal cancer tissues was decreased compared to their normal mucosal tissue.3,Then,we explored the effect of GPR12 on the function of hypopharyngeal cancer,laryngeal cancer and esophageal cancer.In order to study the effect of GPR12 on cell migration,we adopted some assays.The wound healing assay and transwell assay results showed that GPR12 can significantly inhibit the migration ability of hypopharyngeal carcinoma Fadu cells,laryngeal carcinoma Hep2 cells and esophageal carcinoma Eca109 cells.In order to study the effect of GPR12 on cell apoptosis,we performed flow cytometry apoptosis assay and Caspase-Glo(?)3/7 assay.Flow cytometry results showed that high expression of GPR12 could significantly promote the apoptosis of Fadu,Hep2 and Eca109 cells.Caspase-Glo(?)3/7 assay results showed that overexpression of GPR12 could increase the activity of Caspase-Glo(?)3/7 in Fadu,Hep2 and Eca109 cells,which leads to apoptosis of Fadu,Hep2 and Eca109 cells.In order to study the effect of GPR12 on the cell cycles and proliferation of hypopharyngeal carcinoma,laryngeal carcinoma and esophageal cancer cells,we used flow cytometry to determine the effect of GPR12 on the cell cycle,and used a CCK-8 kit assay to explore the effect of GPR12 on cell proliferation.The results of flow cytometry showed that GPR12 had no effect on the cell cycles of Fadu,Hep2 and Eca109.The results of CCK-8 kit assay showed that GPR12 had no effect on the proliferation of Fadu,Hep2 and Eca109 cells.4,In order to explore the mechanism,we used GSEA database analysis to study the relationship between GPR12 and the related tumor hallmark gene sets.We found that GPR12 was negatively correlated with several hallmark gene sets that were closely related to tumor progression of cells migration and apoptosis such as EMT,MTORC1,MYC,DNA repair and TNFa proteins.5,Therefore,we further study the functional mechanism of GPR12.According to western blot experiments,we found that GPR12 could increase the expression of EMT-related proteins E-cadherin and α-catenin in hypopharyngeal cancer,laryngeal cancer and esophageal cancer cells,thereby inhibiting cell migration.Meanwhile,GPR12 could increase the expression of activated caspase-7 protein in hypopharyngeal cancer,laryngeal cancer and esophageal cancer cells,thereby promoting cell apoptosis.ConclusionIn summary,GPR12 induces apoptosis by activating caspase-7,and inhibits migration by promoting the expression of EMT-related proteins E-cadherin and α-catenin in hypopharyngeal carcinoma,laryngeal carcinoma and esophageal carcinoma cells.Our results show that GPR12 as a potential tumor factor mediates the migration and apoptosis of hypopharyngeal cancer,laryngeal cancer and esophageal cancer cells and provide new molecular targets for the treatment of hypopharyngeal cancer,laryngeal cancer and esophageal cancer.