| BackgroundPancreatic cancer is a highly malignant gastrointestinal tumor with high recurrence rate,high metastasis rate and poor prognosis.It is predicted to be the third leading cause of death in the near future.Because the lack of effective early diagnosis methods,most pancreatic cancer patients are in an advanced stage when they are diagnosed,and less than 20% of patients with pancreatic cancer are suitable for surgical treatment.Chemotherapy is an important treatment for pancreatic cancer,but chemotherapy resistance has become one of the urgent problems that need to be solved in the pancreatic cancer treatment.In the past decade,although enormous efforts have been made to identify targets for drug discovery and many combination therapies including targeted therapy,immunotherapy have been developed,less progress has been achieved in the treatment of pancreatic cancer,the 5-year survival rate remains at10%.Therefore,exploring the key genes that regulate the occurrence and development of pancreatic cancer will provide a theoretical basis for the development of more effective treatments for pancreatic cancer.G protein-coupled receptor class C group 5 member A(GPRC5A),also known as retinoic acid-inducible gene 3(RAI3)or retinoic acid-inducible gene 1(RAIG1),is a member of the GPCRs family.Recently,studies have found that GPRC5 A is low expressed in lung cancer,head and neck squamous cell carcinoma and oral squamous cell carcinoma,and acts as a tumor suppressor.Overexpression of GPRC5 A inhibits the progression of the above tumors.However,GPRC5 A is high expression and promotes tumor occurrence and metastasis in gastric cancer,ovarian cancer and prostate cancer.At present,domestic and foreign studies mainly explore that GPRC5 A may act as an oncogene in pancreatic cancer and is related to gemcitabine resistance,but its specific mechanism of promoting cancer is incompletely clear.Therefore,finding the key molecules downstream of GPRC5 A and their mechanism in pancreatic cancer is meaningful.Neuropeptide S receptor 1(NPSR1),also known as orphan G protein-coupled receptor 154(GPR154)or asthma susceptibility G protein-coupled receptor(GPRA),is a member of the GPCRs family.There are few studies on NPSR1 in tumors,only NPSR1 is high expression in neuroendocrine tumors and lung adenocarcinoma,and can be used as a biomarker.In addition,NPSR1 may promote the malignant biological behavior of pancreatic cancer cells.Therefore,NPSR1 may play an oncogenic role in pancreatic cancer,but the specific molecular mechanism for the regulation of tumor progression remains unclear and no related research reported.Therefore,exploring the clinical significance,the role and molecular mechanism of NPSR1 in pancreatic cancer is expected to provide new directions and ideas for clinical treatment,prognosis judgment and possible intervention targets of pancreatic cancer.Part I Upregulated GPRC5 A disrupting the Hippo pathway promotes the proliferation and migration of pancreatic cancer cells via the c AMP?CREB axis ObjectiveTo explore the effect of GPRC5 A on the proliferation and migration of pancreatic cancer cells,and further molecular mechanism.MethodsGPRC5A expression in pancreatic cancer tissues and its correlation with clinicopathological features and survival prognosis of patients with pancreatic cancer were evaluated using bioinformatics.The expression of GPRC5 A was evaluated and measured using real-time quantitative PCR,immunohistochemistry and western blotting in pancreatic cancer tissues and cells.The biological function of GPRC5 A in pancreatic cancer was assessed using CCK-8,colony formation,wound-healing,transwell assays,and the xenograft model of pancreatic cancer.Using RNA sequencing,real-time quantitative PCR,western blotting and the cell recovery assays to explore the regulation of GPRC5 A on Hippo signaling pathway.The regulation effect of GPRC5 A on c AMP-CREB signaling pathway was investigated by real-time quantitative PCR and western blotting.ResultsGPRC5A expression was increased in pancreatic cancer tissues and cells,and high GPRC5 A expression was significantly correlated with clinicopathological features and poor prognosis of pancreatic cancer patients.Knockdown of GPRC5 A significantly reduced the proliferation and migration of pancreatic cancer cells,and disruption of GPRC5 A expression inhibited tumor growth in vivo.In terms of mechanism,GPRC5 A and YAP1 and their downstream target genes are significantly positively correlated in pancreatic cancer.Knockdown or overexpression of GPRC5 A prominently decreased or increased,respectively,the expression of YAP1 and its target genes,CYR61,cyclin D1,c-Myc and CTGF.Regulation of GPRC5 A expression did not impact on MST1/2,LATS1/2 and its phosphorylated states,which is the upstream kinase that regulates the YAP1 activity.In addition,YAP1 and p-YAP1 changed synchronously without a significant change in the ratio between YAP1 and p-YAP1 based on the regulation of GPRC5 A expression.Moreover,GPRC5 A positively regulated YAP1,CYR61,cyclin D1,c-Myc and CTGF expression at the transcriptional level,as evidenced by overexpression and knockdown of GPRC5 A in pancreatic cancer cells.Taken together,our results show that GPRC5 A dysregulates Hippo signaling by regulating YAP1 transcription.Then,to further determine the role of YAP1 in GPRC5A-mediated function in pancreatic cancer,we upregulated YAP1 using YAP1 plasmid with or without downregulation of GPRC5 A in pancreatic cancer cells.Upregulating YAP1 only increased its expression without influencing GPRC5 A expression in pancreatic cancer cells.In addition,YAP1 overexpression partially counteracted the effects of GPRC5 A knockdown.Interestingly,we downregulated YAP1 using si RNA with or without GPRC5 A overexpression in pancreatic cancer cells.Disrupting YAP1 only reduced its expression without influencing GPRC5 A expression in pancreatic cancer cells.In addition,YAP1 deficiency partially counteracted the effects of GPRC5 A upregulation of YAP1 and its target gene CYR61.Furthermore,the influence of GPRC5 A on promoting the proliferation and migration of pancreatic cancer cells was partially reversed by YAP1 deficiency in pancreatic cancer cells,which was verified by cell function assays.In summary,our findings demonstrated that inhibiting YAP1 was beneficial in disrupting the adverse effects of GPRC5 A on pancreatic cancer cells.Finally,knockdown or overexpression of GPRC5 A could decrease or increase the m RNA expression of c AMP.Moreover,silencing or overexpressing GPRC5 A drastically decreased or increased the phosphorylation of CREB without significantly affecting total CREB expression.Intriguingly,we observed that nuclear localization of phosphorylated CREB was upregulated and cytoplasmic phosphorylated CREB expression was downregulated upon GPRC5 A overexpression.Conversely,GPRC5 A knockdown decreased nuclear phosphorylated CREB levels and promoted cytoplasmic phosphorylated CREB expression.Taken together,these data suggested that GPRC5 A disrupted the Hippo-YAP1 pathway through c AMP-CREB signaling.ConclusionsGPRC5A promotes pancreatic cancer progression by regulating the expression of the core protein YAP1 of the Hippo pathway.These findings reveal the crosstalk between GPCRC5 A and Hippo pathways,which is beneficial to the in-depth understanding of pancreatic cancer progression.Part II NPSR1 promotes the proliferation and invasion of pancreatic cancer cells by regulating the MAPK signaling pathwayObjective To explore the effect of NPSR1 on the malignant biological behavior of pancreatic cancer cells and the molecular mechanism of NPSR1 promoting pancreatic cancer progression.Methods NPSR1 expression in pancreatic cancer tissues and its correlation with survival prognosis of patients with pancreatic cancer were evaluated using bioinformatics.The expression of NPSR1 was evaluated and measured using real-time quantitative PCR,immunohistochemistry and western blotting in pancreatic cancer tissues and cells.The biological function of NPSR1 in pancreatic cancer was assessed using CCK-8,colony formation,transwell assays,and the xenograft model of pancreatic cancer.Using realtime quantitative PCR,western blotting and the cell recovery assays to explore the regulation of NPSR1 on MAPK signaling pathway.Results NPSR1 expression was increased in pancreatic cancer tissues and cells,and high GPRC5 A expression was significantly correlated with poor prognosis of pancreatic cancer patients.Knockdown or overexpression of NPSR1 significantly reduced or increased the proliferation and invasion of pancreatic cancer cells,and overexpression of NPSR1 promoted tumor growth in vivo.In terms of mechanism,knockdown or overexpression of NPSR1 prominently decreased or increased,respectively,the expression of N-cadherin and Vimentin and increased or decreased the expression of E-cadherin.Therefore,NPSR1 promoted the progression of pancreatic cancer by affecting the EMT process.Then,previous studies showed NPSR1 affects the development of tumors by activating the JNK/ERK MAPK pathway.Silencing or overexpressing NPSR1 drastically decreased or increased the phosphorylation of ERK1/2 and JNK without significantly affecting total ERK1/2 and JNK expression.Therefore,NPSR1 activated the JNK/ERK MAPK signaling pathway in pancreatic cancer.Finally,since EMT and MAPK signaling pathways were activated by NPSR1,we explored whether NPSR1 affects EMT processes by regulating JNK/ERK MAPK signaling pathways.We used JNK inhibitor(SP600125)and ERK1/2 inhibitor(SCH772984)respectively with overexpression of NPSR1 in pancreatic cancer cells,and found that overexpression of NPSR1 could upregulate the expression of Ncadherin and Vimentin and downregulate the expression of E-cadherin.However,the effect of NPSR1 on promoting the expression of N-cadherin and Vimentin and inhibiting the expression of E-cadherin could be partially reversed by JNK inhibitor and ERK1/2 inhibitor,respectively.Furthermore,the influence of overexpression of NPSR1 on promoting the proliferation and invasion of pancreatic cancer cells was partially reversed by SP600125 or SCH772984 in pancreatic cancer cells,which was verified by cell function assays.Conclusions To sum up,NPSR1 may promote the proliferation and invasion of pancreatic cancer cells via MAPK signaling pathway.Our results suggest that NPSR1 may be a prognostic marker and potential therapeutic target for pancreatic cancer. |
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