| BackgroundAt present,platinum resistance in non-small cell lung cancer(NSCLC)is still the main cause of poor prognosis in most patients with NSCLC.However,the specific mechanism of platinum resistance in non-small cell lung cancer has not been fully understood.Exosomes and immune regulation may play important roles in platinum resistance.Firstly,exosomes are nano vesicles secreted by cells carrying bioactive substances.Based on previous studies,we assumed that exosomes may transfer drug resistance through APE1 or micro RNA.Secondly,the combination of programmed cell death receptor ligand 1(PD-L1)and programmed cell death receptor(PD-1),the key molecule of immune checkpoint on tumor cells,can inhibit the immune response,and the immune state of the body is closely related to the efficacy of chemotherapy.Therefore,we speculate that PD-L1 SNP may also affect the efficacy of platinum based chemotherapy.The present study will explore the mechanism of platinum resistance in NSCLC from the above two aspects,so as to provide theoretical basis for comprehensive evaluation of the effectiveness of platinum chemotherapy in patients with NSCLC,and take corresponding measures to improve the treatment effect and prognosis.Methods1.Exosomes were collected by high-speed centrifugation or exosome extraction kit.After the exosomes were stained with PKH26,the cells were observed by confocal laser scanning.The expression of GST-APE1 protein was observed after GST-APE1 protein incubated with A549 cells.CCK-8 assay and flow cytometry were used to detect the viability of cells in different concentrations of cisplatin.Transwell assay was used to evaluate cell invasion and migration.After knockdown of APE1 in A549 cells,A549 cells incubated with different groups of exosomes and then treated with APE1 functional inhibitor then survival-H2 AX protein were observed.A total of136 NSCLC patients who received platinum chemotherapy were selected to extract and identify the plasma exosome.Western blot and ELISA were used to detect the expression of APE1 in plasma exosomes,and immunohistochemistry was used to detect the expression of APE1 in tissues.According to Response Evaluation Criteria in Solid Tumors,the patients were divided into platinum chemotherapy responder group(CR+PR)and platinum chemotherapy non-responder group(SD+PD),then the relationship between APE1 expression level and treatment sensitivity was explored.2.Micro RNA microarray was used to detect the changes of micro RNA in exosomes treated with cisplatin.A549 cells were transfected with mi R-1273 a mimic,inhibitor or negative control to construct cells with different mi R-1273 a expression levels.CCK-8 assay and flow cytometry were used to detect the effect of mi R-1273 a on cell survival ability in cisplatin.A549 cells were incubated with exosomes with high mi R-1273 a expression to study the effect of overexpression of mi R-1273 a on cell viability in cisplatin.The target gene of mi R-1273 a was predicted by bioinformatics method and verified by Western blot and q PCR.A total of 85 patients with NSCLC treated with platinum chemotherapy were selected for clinical evaluation.The level of micro RNA in plasma exosomes was detected by RT-q PCR,and plasma SDCBP or APE1 level were measured by ELSIA.3.Blood samples were collected from 281 patients with NSCLC treated with platinum chemotherapy and the DNA from blood was extracted.The control group consisted of 251 volunteers who received health check-ups at the same time and did not suffer from cancer or any other serious diseases.The SNPs were genotyped using MALDI-TOF MS in a Mass ARRAY system.Chi-square test was used to compare the genotype distribution of different groups.Kaplan-Meier analysis and Cox univariate/multivariate survival analysis were used to evaluate the prognosis of patients with different genotypes.Results1.NSCLC derived exosomes were successfully collected.After PKH26 staining,the stained exosomes were observed in the receptor cells.When A549 cells were co-incubated with exosomes,the sensitivity of A549 cells to cisplatin was decreased,the apoptosis rate was decreased,and the proliferation,migration and invasion ability of A549 cells were enhanced.After treatment with cisplatin,the expression of APE1 increased both in A549 cells and exosomes.After incubation with exosomes with high expression of APE1,the sensitivity of cells to cisplatin decreased,the apoptosis rate decreased,and the proliferation,migration and invasion of cells were enhanced.The base excision repair function of APE1 plays a major role in the exosome APE1 mediated changes in cell sensitivity to cisplatin.In clinical samples,APE1 in exosomes is the main form of APE1 in peripheral blood.The expression of APE1 in exosomes of NSCLC patients was higher than that of healthy controls.The level of APE1 in plasma of patients who did not respond to platinum chemotherapy was significantly higher than that in patients who responded to platinum chemotherapy.2.Compared with the control group,276 mi RNAs were more than 2-fold change in exosomes stimulated by cisplatin,and mi R-1273 a was found to be the most prominently downregulated mi RNA in cisplatin-treated exosomes.Overexpression of mi R-1273 a enhanced the sensitivity of A549 cells to cisplatin,and the exosomal delivery of mi R-1273 a also enhanced the sensitivity of A549 cells to cisplatin.SDCBP Syndecan binding protein may be one of the targets of mi R-1273 a.The level of mi R-1273 a in plasma of patients in non-responder group was significantly lower than that of patients in responder group.The results of the cooperative project showed that 61 mi RNAs in exosomes changed significantly after APE1 knockdown in A549 cells.The changes of mi R-130 b and mi R-200 c may be related to the efficacy of platinum based chemotherapy in NSCLC.3.The frequency of G allele of PD-L1 rs7866740 in NSCLC patients was significantly higher than that in controls(P = 0.001).The risk of NSCLC in individuals with G allele was significantly higher than that in individuals with C allele(OR = 3.532,95%CI =1.232-10.129).Kaplan Meier analysis showed that PFS and OS of PD-L1 rs2890658 CA +AA genotype were significantly lower than those of CC genotype(P < 0.05),and the OS of PD-L1 rs822336 GC + CC was significantly lower than that of GG genotype(P < 0.05).Cox survival analysis showed that PD-L1 rs2890658 CA + AA was a risk factor for PFS and OS in NSCLC patients(PFS: adjusted HR = 1.367,95% CI = 1.0-1.8,P = 0.038;OS: adjusted HR =1.402,95%CI = 1.0-1.9,P = 0.026).PD-L1 rs822336 GC + CC was a risk factor for OS(adjusted HR = 1.393,95%CI = 1.1-1.8,P = 0.021),but there was no significant correlation with PFS.Conclusion1.The expression of exosomal APE1 increased after cisplatin treatment.The exosomes with high expression of APE1 can reduce the sensitivity of NSCLC to platinum drugs.2.The base excision repair function of APE1 may play a key role in platinum chemoresistance mediated exosomal APE1.3.Plasma exosomal APE1 expression was related to the efficacy of platinum based chemotherapy in patients with NSCLC.4.The decreased of exosomal mi R-1273 a was related to the decrease of platinum sensitivity of receptor cells.5.Plasma exosomal mi R-1273 a expression levels in patients with NSCLC are associated with the efficacy of platinum based chemotherapy.6.APE1 may promote platinum resistance by regulating the expression levels of mi R-130 b and mi R-200 c in exosomes.7.PD-L1 rs7866740 is associated with NSCLC susceptibility.PD-L1 rs2890658 and rs822336 are associated with the prognosis of NSCLC. |
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