Mechanism Of Circ＿0006988 In Gefitinib Resistance Of Lung Adenocarcinomar

Objective:Lung cancer is one of the most common malignant tumors,and its incidence rate and mortality are increasing year by year.The most common type of lung cancer is lung adenocarcinoma.Targeted therapy is currently one of the most common treatment methods for advanced non-small cell lung cancer.Activation of epidermal growth factor receptor(EGFR)can promote lung cancer cell differentiation and proliferation,while overexpression of EGFR can lead to poor prognosis in lung cancer.Gefitinib is one of the most commonly used drugs for targeted therapy,but it develops acquired resistance to EGFR-TKIs after a period of use.The expression changes of tumor related genes are closely related to the drug resistance mechanism of tumors.It is of great significance to explore the pathogenesis of NSCLC and the mechanism of gefitinib resistance,and find more effective molecular therapeutic targets to improve gefitinib resistance.circular RNA(circ RNA)is involved in the biological processes of tumor occurrence,progression,and drug resistance formation,and is an extremely important molecular target in tumor targeted therapy.circ RNA can interfere with the occurrence and progression of tumors.micro RNA(miRNA)can regulate gene expression horizontally after transcription.circ RNA mainly regulates the expression of downstream target genes of miRNA by binding to miRNA,thereby intervening in the growth,invasion,apoptosis and other processes of tumor cells.circ＿0006988 is a gene regulatory factor closely related to tumor,which plays a vital role in the occurrence and progress of tumor,and may be involved in the process of tumor drug resistance.The purpose of this study is to explore the specific effect of circ＿0006988 on gefitinib resistance in PC-9/GR,A549 cells and its related regulatory mechanism on the downstream.Methods:Part 1Real time quantitative polymerase chain reaction(RQ-PCR)was used to detect circ＿0006988 in human lung adenocarcinoma tissues and cells from patients with gefitinib resistance,non gefitinib resistant lung adenocarcinoma tissues and cells,PC-9 cells,PC-9/GR cells,A549 cells,A549/GR cells,and BEAS-2B cells(human normal bronchial epithelial cells)using real-time quantitative PCR/real-time quantitative PCR.The specific expression differences of circ＿0006988.Regulation of circ＿0006988 in lung adenocarcinoma cells(PC-9 cells,A549 cells)and gefitinib resistant cells(PC-9/GR cells,A549/GR cells)using cell transfection circ＿0006988expression level,and then treated each group of cells with gefitinib.Apoptosis was detected using flow cytometry(FCM),cell clones were detected using plate cell cloning assay,migration and invasion ability of each group of cells was detected using Transwell assay,glycolysis of each group of cells was detected using 2-NBDG,and expression levels of AKT,PTEN,p-AKT,and mTOR proteins in cells were detected using Western Blot(WB)method.Part 2Using the Circinteractome database to predict complementary binding sites between circ＿0006988 and miR-491-5p,and its targeting relationship was verified using dual luciferase reporter gene assay and RNA binding protein immunoprecipitation(RIP)assay.Detect the effect of upregulating or downregulating circ＿0006988 on the expression level of miR-491-5p in PC-9 cells,PC-9/GR,A549 cells,and A549/GR cells using PCR method.The effects of miR-491-5p on circ＿0006988 regulated cell apoptosis,cloning,migration,invasion,glycolysis,and related protein expression levels were detected by cell transfection and treatment with gefitinib on lung adenocarcinoma cells and gefitinib resistant cells in each group.Part 3It is predicted by ENCORI software that miR-491-5p and mitogen-activated protein kinase kinase 3(MAP3K3)have complementary binding sites at the 3’ UTR end,and the targeting relationship between them is verified by luciferase reporter gene system and RNA pull-down experiment.Detect the effect of downregulating or upregulating miR-491-5p on MAP3K3 expression in PC-9 cells,PC-9/GR cells,A549 cells,and A549/GR cells using PCR and WB methods.The effects of MAP3K3 on miR-491-5p regulation of cell apoptosis,cloning,migration,invasion,glycolysis,and related protein expression levels were detected by cell transfection and treatment with gefitinib on lung adenocarcinoma cells and gefitinib resistant cells in each group.Use WB method to detect and analyze the effect of regulating circ＿0006988 on MAP3K3 expression in lung cancer cells and gefitinib resistant cells.Part 4By regulating the levels of MAP3K3 and treating lung adenocarcinoma cells and their gefitinib resistant cells with gefitinib,each group of cells was treated with gefitinib to detect apoptosis,cloning,migration,invasion,glycolysis,and related protein expression levels.Using PCR method to detect the expression levels of MAP3K3 messenger RNA(mRNA)and mTOR mRNA in lung adenocarcinoma tissues of non gefitinib resistant patients and gefitinib resistant patients,and to calculate and analyze the correlation between mTOR and MAP3K3 in lung adenocarcinoma tissues.Use immunoprecipitation(IP)assay to detect the interaction between MAP3K3 and PTEN in PC-9/GR cells and A549/GR cells.Measure and analyze the effect of downregulating MAP3K3 on PTEN ubiquitination degradation in PC-9/GR cells and A549/GR cells using WB method.PC-9/GR cells and A549/GR cells downregulated by MAP3K3 were transplanted subcutaneously into nude mice,and treated with gefitinib.The weight and growth volume of the transplanted tumor were observed,stained with HE staining and TUNEL staining.The degree of cell apoptosis in the transplanted tumor tissue was observed,and the expression level of mTOR protein in the transplanted tumor tissue was detected using WB staining.Results:Part 11.The expression level of circ＿0006988 in human lung adenocarcinoma of patients with gefitinib resistance was significantly higher than that of circ＿0006988 in human lung adenocarcinoma of patients with gefitinib resistance(***P < 0.001).Compared with BEAS-2B cells,the level of circ＿0006988 in A549 cells is higher(*P<0.05),and the level of circ＿0006988 in PC-9 cells is higher(**P< 0.01).The expression level of circ＿0006988 in gefitinib-resistant human lung adenocarcinoma cell line PC-9/GR was significantly higher than that in human lung adenocarcinoma cell line PC-9(P<0.05).The expression level of circ＿0006988 in human lung adenocarcinoma cell line A549/GR with gefitinib resistance was significantly higher than that in human lung adenocarcinoma cell line A549(P<0.05).2.Up-regulation of circ＿0006988 could inhibit the apoptosis of PC-9 cells treated with gefitinib,improve the formation,migration and invasion ability of cell clones,promote the uptake of glucose and the production of lactic acid,promote the expression of p-AKT and mTOR proteins,and inhibit the expression of PTEN proteins.3.Down-regulation of circ＿0006988 can promote the apoptosis of PC-9/GR cells treated with gefitinib,inhibit cell formation,migration and invasion,inhibit the uptake of glucose and the production of lactic acid,inhibit the expression of p-AKT and mTOR proteins,and promote the expression of PTEN protein.Part 21.Luciferase Reporter Assay and RIP experiments proved that circ＿0006988and miR-491-5p were mutually targeted.2.Up-regulation of circ＿0006988 inhibited the expression of miR-491-5p in PC-9 cells.And down-regulation of circ＿0006988 promoted the expression of miR-491-5p in PC-9/GR cells.3.miR-491-5p mimics could reverse the effects of circ＿0006988 up-regulation on apoptosis,cloning,migration,invasion,glucose uptake,lactate content and protein expression of PTEN,p-AKT and mTOR in PC-9 cells treated with gefitinib.miR-491-5p inhibitor can reverse the effects of circ＿0006988down-regulation on apoptosis,cloning,migration,invasion,glucose uptake,lactate content and protein expression of PTEN,p-AKT and mTOR in PC-9 cells treated with gefitinib.Part 31.Luciferase Reporter Assay and RNA sedimentation experiments proved that miR-491-5p and MAP3K3 were mutually targeted.2.Down-regulation of miR-491-5p promoted the expression of MAP3K3 in PC-9 cells.Meamwhile,up-regulation of miR-491-5p inhibited the expression of MAP3K3 in PC-9/GR cells.3.MAP3K3 siRNA could reverse the effects of down-regulation of miR-491-5p on apoptosis,cloning,migration,invasion,glucose uptake,lactate content and protein expression of PTEN,p-AKT and mTOR in PC-9 cells treated with gefitinib.pc DNA-MAP3K3 could reverse the effects of up-regulation of miR-491-5p on apoptosis,cloning,migration,invasion,glucose uptake,lactate content and protein expression of PTEN,p-AKT and mTOR in PC-9/GR cells treated with gefitinib.4.miR-491-5p mimics reversed the up-regulation of circ＿0006988 and promoted MAP3K3 expression in PC-9 cells.miR-491-5p inhibitor reversed the down-regulation of circ＿0006988 in inhibiting MAP3K3 expression in PC-9/GR cells.Part 41.Up-regulation of MAP3K3 inhibited apoptosis of PC-9 cells treated with gefitinib,promoted glucose uptake and lactate production,promoted mTOR and p-AKT protein expression,while inhibited PTEN protein expression;Down-regulation of MAP3K3 promoted the apoptosis of PC-9/GR cells treated with gefitinib,inhibited glucose uptake and lactate production,inhibited the protein expression of p-AKT and mTOR and promoted the protein expression of PTEN.2.The expression levels of MAP3K3 mRNA and mTOR mRNA in gefitinib-resistant non-small cell lung cancer tissues were higher than those in gefitinib-resistant tissues,and the expression levels of MAP3K3 mRNA and mTOR mRNA were positively correlated(R=0.7298,*** P<0.001).3.MAP3K3 and PTEN interact,and down-regulation of MAP3K3 inhibits the ubiquitination degradation of PTEN.4.After down-regulation of MAP3K3,the growth volume and weight of PC-9/GR cell xenografts in nude mice were markedly decreased,the apoptosis of xenografts was markedly increased,and the expression level of mTOR protein was markedly decreased.Conclusions:1.circ＿0006988 was highly expressed in the gefitinib-resistant human lung adenocarcinoma tissue and cells,PC-9/GR cells and A549/GR cells.2.circ＿0006988 targeting negative regulation miR-491-5p.miR-491-5p targeting negative regulation MAP3K3.circ＿0006988 regulates the expression of MAP3K3 through sponge adsorption of miR-491-5p,thereby exerting the function of regulating PC-9/GR cells and A549/GR cells.Reducing circ＿0006988 promotes the apoptosis of PC-9/GR cells and A549/GR cells,inhibits the invasion,migration,cloning and glycolysis of cells,promotes the expression of PTEN protein,inhibits the expression of p-AKT and mTOR proteins,inhibits the gefitinib resistance of cells,and the growth of nude mice transplanted tumors.3.The mechanism of circ＿0006988 influencing gefitinib resistance in PC-9/GR cells and A549/GR cells is related to the regulation of PTEN/AKT/mTOR signal pathway by miR-491-5p/MAP3K3 gene.