| Objective: Non-small cell lung cancer(NSCLC)as a common malignant tumor in the clinic.standard treatments of NSCLC is with.However,due to the emergence of cisplatin resistance,the treatment effect of cisplatin chemotherapy is not very satisfactory.Tumor resistance is related to changes in gene expression,and finding effective therapeutic targets to improve NSCLC cisplatin sensitivity is crucial.circular RNA(circRNA)is a special RNA with no 5'and 3'ends,which are connected end to end to form a closed loop,and this circular combination can make circRNA resistant to exonuclease R(Nase R),so circRNA in tissues expression has more excellent stability.circRNA expression is characterized of specificity and richness,and it has an important role with the occurrence of tumors drug resistance.circRNA may be a target for tumor treatment.The mechanism by which circRNA affects tumor progression is complex.It can affect the expression of downstream genes by combining with micro RNA(miRNA),thereby regulating the biological behaviors of cell growth,apoptosis,and invasion.It is known that circCRIM1 is a tumor-related regulatory factor,which plays a role in the progression of tumors such as nasopharyngeal carcinoma and participates in the development of tumor resistance.The purpose of this study was to investigate the effect of circCRIM1 on A549/DDP cell resistance to cisplatin and downstream regulatory mechanisms.Methods: 1.Quantitative real time polymerase chain reaction(q RT-PCR)was used to detect the difference in A549 and A549/DDP cells of circCRIM1 expression,through cell transfection to up or down regulate circCRIM1 expression in A549 and A549/DDP,given cisplatin treatment,and use flow cytometry to detect cell apoptosis Death,plate cloning test to detect cell clone formation ability,Transwell chamber was used to detect cell migration and invasion ability,2-NBDG flow cytometry was used tdetect glucose uptake,biochemical method was used to detect lactic acid content,Western blot method was used to detect phosphatase and p-AKT,tensin homolog deleted on chromosome 10(PTEN),hexokinase 2(HK2),protein kinase B(AKT)protein expression.2.Using bioinformatics software(circbank)to predict that circCRIM1 and miR-507 had complementary binding sites,luciferase reporter system and RNA binding protein immunoprecipitation(RIP)experiment to verify the targeting relationship;q RT-PCR detection up or down regulation of circCRIM1 on miR-507 expression of A549,A549/DDP cells;co-transfected circCRIM1 overexpression vector,miR-507 mimics or circCRIM1 si RNA,miR-507 inhibitor into A549,A549/DDP cells,treated with cisplatin,flow cytometry was used to detect apoptosis,the cloning ability of the cells was detected by plate cloning experiment,the cell migration and invasion ability was detected by Transwell cell,glucose uptake was detected by the2-NBDG flow cytometry method,lactate content was detected by the biochemical method,and the the expression of PTEN,AKT,p-AKT and HK2 protein was detected by Western blot.3.Bioinformatics software(targetscan)predicted that miR-507 and Tripartite motif-containing59(TRIM59)had complementary binding sites at the 3? UTR end,luciferase reporter system and RNA pull-down experiments verified the targeting relationship;q RT-PCR and Western blot detected the expression of TRIM59 in down-regulated or up-regulate in A549,A549/DDP cells;co-transfected miR-507 inhibitor,TRIM59 si RNA or miR-507 mimics,pc DNA-TRIM59 into A549,A549/DDP cells,and then treated with cisplatin,flow cytometry was used to detect apoptosis,the cloning ability of the cells was detected by plate cloning experiment,the cell migration and invasion ability was detected by Transwell cell,glucose uptake was detected by the2-NBDG flow cytometry method,lactate content was detected by the biochemical method,and the the expression of PTEN,AKT,p-AKT and HK2 protein was detected by Western blot;the expression of TRIM59 in A549,A549/DDP cells of up-regulation or down-regulation of circCRIM1 and co-transfected circCRIM1 overexpression vector,miR-507 mimics or circCRIM1 si RNA,miR-507 inhibitor was detected by Western blot.4.Increased or decreased the expression level of TRIM59 in A549,A549/DDP cells,given cisplatin treatment,flow cytometry to detect apoptosis,2-NBDG flow cytometry to detect glucose uptake,biochemical method to detect lactic acid content,and the the expression of PTEN,AKT,p-AKT and HK2 protein was detected by Western blot.5.Detected the expression of TRIM59 m RNA and HK2 m RNA in cisplatin-resistant(NR)and cisplatin-resistant(R)non-small cell lung cancer tissues by q RT-PCR method,and then analyzed the correlation between the two;IP test detection the interaction between TRIM59 and PTEN in A549/DDP cells;Western blot analysis of the effect of downregulation of TRIM59 on the ubiquitination and degradation of PTEN in A549/DDP cells.6.A549/DDP cells with down-regulated TRIM59 was inoculateed into the nude mice,and then given cisplatin treatment,observed the growth volume and weight of the transplanted tumor in nude mice.Observed the apoptosis in the transplanted tumor tissue by TUNEL staining and HE staining.Detected the HK2 protein expression in tumor tissue by Western blot.Results:1.circCRIM1 expression in A549/DDP cells was higher than that of lung cancer cells A549(P<0.05);up-regulation of circCRIM1 could inhibit the apoptosis of cisplatin-treated A549 cells,and improved cell clone formation,migration,and invasion ability,promoted glucose uptake and lactate production,promoted p-AKT,HK2 protein expression,inhibited PTEN protein expression;down-regulate circCRIM1 to promote cisplatin A549/DDP cell apoptosis,inhibited cell clonal formation,migration,invasion ability,inhibit cellular glucose uptake and lactate production,inhibited p-AKT,HK2 protein expression,promoted PTEN protein expression.2.Luciferase reporter system and RIP experiments proved that circCRIM1 and miR-507 were mutually targeted;up-regulating circCRIM1 inhibited the expression of miR-507 in A549 cells,and down-regulating circCRIM1 promoted the expression of miR-507 in A549/DDP cells;miR-507 mimics could reverse the effects of upregulation of circCRIM1 on cisplatin A549 cell apoptosis,cloning,migration,invasion,glucose uptake,lactate content,and the expression of PTEN,p-AKT,HK2 protein;miR-507 inhibitor could reverse the downregulation of circCRIM1 on cisplatin A549 cell apoptosis,cloning,migration,invasion,glucose uptake,lactate content and the expression of PTEN,p-AKT,HK2 protein.3.Luciferase reporter system and RNA pull-down experiments proved that miR-507 and TRIM59 were mutually targeted;down-regulation of miR-507 promoted TRIM59 expression in A549 cells,and up-regulation of miR-507 inhibited TRIM59 expression in A549/DDP cells;TRIM59 si RNA could reverse the effects of miR-507 on apoptosis,cloning,migration,invasion,glucose uptake,lactate content,and PTEN,p-AKT,HK2 protein expression of A549 cells affected by miR-507 on cisplatin;pc DNA-TRIM59 could reverse the upregulation of miR-507 cisplatin-induced A549/DDP cell apoptosis,cloning,migration,invasion,glucose uptake,lactate content,and PTEN,p-AKT,and HK2 protein expression;miR-507 mimics reversed upregulation of circCRIM1 to promote TRIM59 expression in A549 cells;miR-507 inhibitor reversed down-regulates the effect of circCRIM1 on TRIM59 expression in A549/DDP cells.4.Up-regulation of TRIM59 inhibited cisplatin-induced A549 cell apoptosis,promoted glucose uptake and lactate production,promoted p-AKT and HK2 protein expression,and inhibited PTEN protein expression;down-regulates TRIM59 promoted cisplatin-induced A549/DDP cell apoptosis,inhibited glucose uptake and lactic acid production,inhibited p-AKT and HK2 protein expression,and promoted PTEN protein expression.5.The expression levels of TRIM59 m RNA and HK2 m RNA in cisplatin-resistant non-small cell lung cancer tissues were higher than that of cisplatin-resistant tissues,and the expression levels of the TRIM59 m RNA and HK2 m RNA had positively correlated(R=0.7306,P<0.001);There was interaction between TRIM59 and PTEN,and down-regulation of TRIM59 inhibited the ubiquitination degradation of PTEN.6.The growth volume and weight of transplanted tumors of A549/DDP cells with down-regulated TRIM59 in nude mice were significantly reduced,the apoptosis in transplanted tumor tissues increased,and the expression level of HK2 protein decreased.Conclusions: 1.circCRIM1 was highly expressed in A549/DDP cells,down-regulating circCRIM1 promoted cisplatin-induced A549/DDP cell apoptosis,inhibited cell cloning,migration,invasion and glycolysis levels.2.circCRIM1 affected the apoptosis,cloning,migration,invasion and glycolysis of A549/DDP cells by negatively regulate miR-507 with cisplatin.3.Mi R-507 affected the apoptosis,cloning,migration,invasion and glycolysis of A549/DDP cells by negatively regulate TRIM59 with cisplatin.4.circCRIM1 and miR-507 jointly regulated TRIM59 expression.5.Down-regulation of TRIM59 inhibited cisplatin resistance of A549/DDP cells by regulating PTEN/AKT/HK2.6.Down-regulation of TRIM59 inhibited the growth of A549/DDP cells in nude mice. |
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