| According to the World Health Organization(WHO)statistics in 2020,lung cancer is the leading cause of cancer-related death in 60 countries around the World.Lung cancer is the most common tumor in China,and it has become a serious threat to human health and a public safety issue.G protein-coupled receptor(GPCR)is the largest membrane protein family in eukaryotes.Alterations in GPCR gene expression and dysregulation of signal transduction have been recognized as markers of malignancy.GPR37 is a member of GPCR,but its role in tumors,especially in non-small cell lung cancer(NSCLC),is still unknown.(1)Data analysis and clinical verification of GPR37 in NSCLCAt home and abroad there is no such research on the expression of GPR37 and its mechanism of action in NSCLC.We first screened the data in the public database of the Cancer Genome Atlas(TCGA)and found that GPR37 in different tumors had different expressions.The expression of GPR37 in tumor tissues of lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)was significantly higher than that in normal tissues,and the difference was statistically significant(P<0.001).We used the Survival Analysis module of Gene Expression Profiling Interactive Analysis 2(GEPIA2)to analyze the overall survival(OS).In the overall survival analysis,we found that the prognosis of the GPR37 high expression group was worse than the GPR37 low expression group,and the difference was statistically significant(P<0.05).Subsequently,we verified by collecting the information of 20 patients who were clearly diagnosed as NSCLC in our center.We found that the expression of GPR37 in tumor tissues was significantly higher than that in paired adjacent tissues(P<0.001).Through cytological experiments,it was found that GPR37 was highly expressed in NSCLC cell lines,and there was a statistical difference between it and the normal cells group(P<0.001).(2)The effect of GPR37 on the biology of NSCLCIn this study,lentivirus packaging plasmid with overexpression and knockdown of GPR37 were constructed and transfected into A549,H1299 and H460 cells to obtain stable expression of GPR37 overexpression(GPR37 group),empty vector group(Vector group),GPR37 knockdown group(sh GPR37 group)and GPR37 knockdown control group(sh-Mock group).We conducted experiments on the effects of GPR37 on cell function in the above-mentioned different groups.The results showed that,taking A549 cell line as an example,the cell viability of GPR37 group was significantly higher than that of the Vector group under the same conditions according to cell counting kit-8(CCK8)test(P<0.001).The number of cell viability in the sh GPR37 groups were significantly lower than that in the sh-Mock group with a significant difference(P<0.001).Through the clone formation experiment,it was found that the clone formation ability of the GPR37 group was significantly stronger than that of the Vector group with a significant difference(P<0.05).The clone formation ability of the sh GPR37 group was significantly weaker than that of the sh-Mock group with a significant difference(P<0.05).Through the 5-Ethynyl-2'-deoxyuridine(EDU)experiment,it was found that the DNA synthesis ability of the GPR37 group was significantly stronger than that of the Vector group with a significant difference(P<0.01).The DNA synthesis ability of the sh GPR37 group was significantly weaker than that of the sh-Mock group with a significant difference(P<0.05).Through the cell apoptosis rate detection of fluorescence-activated cell sorting(FACS),it was found that the apoptosis rate of GPR37 group was slightly lower than that of the Vector group.Considering that overexpression of GPR37 could reduce the normal apoptosis of cells to a certain extent,but there was no statistically significant difference,while the apoptosis of sh GPR37 group was slightly higher than that of the sh-Mock group,and there was a statistically significant difference(P<0.01).Through cell scratch experiments,it was found that the cell migration ability of the GPR37 group was significantly stronger than that of the Vector group with a significant difference(P<0.001).The cell migration ability of the sh GPR37 group was significantly weaker than that of the sh-Mock group with a significant difference(P<0.01).Through the Transwell experiment,it was found that the invasion ability of the cells in the GPR37 group going through the matrigel and the filter membrane was significantly stronger than that of the Vector group,and there was a statistically significant difference(P<0.001).The invasion ability of sh GPR37 group was significantly weaker than that of the sh-Mock group,and there was a statistically significant difference(P<0.05).We also observed the addition of cisplatin with gradient concentrations from low to high in different groups through the CCK8 experiment,and found that under the action of different concentrations,the cell viability of the GPR37 group was significantly stronger than that of the Vector group.The cell viability of the sh GPR37 group was significantly weaker than that of the sh-Mock group.Through FACS experiments,we observed that the cisplatin at a concentration of 1.0 ug/ml was added to different groups.It was found that the anti-apoptotic ability of the GPR37 group was significantly stronger than that of the Vector group with significant difference(P<0.01).The anti-apoptotic ability of the sh GPR37 group significantly weaker than the sh-Mock group,there was a significant difference(P<0.01).Similar results were obtained for all H1299 and H460 cell lines.The above experiments proved that the overexpression of GPR37 gene could promote the proliferation,migration and invasion of NSCLC cells,could resist apoptosis and reduce the sensitivity of chemotherapeutics,while the knockdown of GPR37 gene could inhibit the proliferation,migration and invasion of NSCLC cells,could promote cell apoptosis and increased the sensitivity of chemotherapy drugs to a certain extent,which might become a new target in the treatment of NSCLC.In vivo experiments,through subcutaneous tumor formation in nude mice,it was found that the tumor volume and weight of the GPR37 group were significantly higher than those of the Vector group(P values were all less than 0.001),while the tumor volume and weight of sh GPR37 were significantly lower than those of the sh-Mock group(P values were all less than 0.001).The results proved that the overexpression of GPR37 could promote the tumorigenesis and growth of NSCLC,and the knockdown of GPR37 gene could inhibit the tumorigenesis and growth of NSCLC.All experiments were repeated three times or more.(3)Research on the mechanism of GPR37 promoting the development of NSCLCFinally,we used GPR37 to study the molecular mechanism of inducing epithelialmesenchymal transitions(EMT),activating the PI3K/Akt/m TOR pathway and the Raf/MEK/ ERK pathway to promote the occurrence and development of tumors.We found that in the A549 cell line,the expression of E-Cadherin in the GPR37 group was decreased compared to the Vector group,while the expression of N-Cadherin and Vimentin was up-regulated.Compared with the sh-Mock group,the expression of E-Cadherin in the sh GPR37 group was up-regulated,while the expression of N-Cadherin and Vimentin was down.The results of H1299 and H460 cell lines were the same as the A549 cell line.It showed that GPR37 promoted the occurrence of EMT and would inhibit the occurrence of EMT in GPR37 knockdown.At the same time,we also studied the effects of GPR37 and PI3K/Akt/m TOR,GPR37 and Raf/MEK/ERK signal transduction pathways.We found that the expressions of P-AKT,P-PI3 K,P-m TOR,P-p70S6 K,and P-EIF4EBP1 in the GPR37 group were significantly higher than those in the Vector group.On the contrary,in the sh GPR37 group,P-AKT,P-PI3 K,P-m TOR,P-p70S6 K,and P-EIF4EBP1 were significantly lower than that of the sh-Mock group,which proved that GPR37 could activate key proteins in the PI3K/Akt/m TOR pathway and promote the pathway active.Similarly,by measuring the expression of key proteins in the Raf/MEK/ERK signal transduction pathway,we found that similar results were obtained in the three cell lines.The expression of P-Raf1,P-MEK1,P-ERK in the GPR37 group was significantly higher than that of the Vector group,and sh GPR37 would significantly decrease the expression of P-Raf1,P-MEK1,and P-ERK comparing to the sh-Mock group.Key proteins were phosphorylated to promote the activation of the pathway.GPR37 knockdown could inhibit the activation of PI3K/Akt/m TOR,Raf/MEK/ERK,and inhibit the occurrence,development of NSCLC.All the above experiments were repeated three times or more.In summary,this study found that the expression of GPR37 in NSCLC patients was significantly higher than that in the general population,and the high expression of GPR37 predicted a poor prognosis.Overexpression of GPR37 gene could promote the proliferation,migration and invasion of NSCLC cells,resist apoptosis,reduce the sensitivity of chemotherapy drugs,promote the growth of NSCLC in vivo,induce EMT,and activate PI3K/Akt/ m TOR and Raf/MEK/ERK signal transduction pathway.GPR37 knockdown could inhibit the proliferation,migration and invasion of NSCLC cells,promote cell apoptosis,increase the sensitivity of chemotherapy drugs to a certain extent,inhibit the growth of NSCLC in vivo,reduce the occurrence of EMT,mainly affect inhibition of PI3K/Akt/m TOR and Raf/MEK/ERK signal transduction pathways.Therefore,these results indicate that GPR37 plays as a tumor promoting role in the occurrence and development of NSCLC.Knockdown of GPR37 could inhibit the occurrence and development of NSCLC.GPR37 may become a potential therapeutic target for the treatment of NSCLC. |
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