Long Noncoding RNA CAR10 Promotes Gefitinib Resistance Via YB-1/PD-L1 Axis In Gefitinib Resistant Lung Adenocarcinoma Cell

Objective:Lung cancer has ranked the most common cause of tumor-related deaths in China.Lung adenocarcinoma(LUAD)is the main pathological subtype of non-small-cell lung cancer(NSCLC)with a poor prognosis.Many lung cancer patients are already in intermediate and advanced phase at diagnosis,and lost the opportunity for operation,while the traditional chemoradiotherapy treatment had no obvious effect.Tyrosine kinase inhibitors(EGFR-TKIs)has significantly improved outcomes in EGFR-mutated lung adenocarcinoma patients.However,acquired drug resistance to EGFR-TKI therapy almost eventually happens,leading to cancer progression.Therefore,it is of a great scientific and clinical significance to have a better understanding of the potential mechanisms of how to improve the sensitivity of lung adenocarcinoma cells to gefitinib.LncRNAs play a importment roles in tumorigenesis and are associated with drug resistance.However,the role and regulatory mechanisms that lncRNAs play in gefitinib resistant lung adenocarcinoma cell have not been fully elucidated.Therefore,the present research was to study the expression of CAR 10 in human lung adenocarcinoma tissues and to investigate its relationship with gefitinib-resistance in lung adenocarcinoma celllines.Methods:1、Real-time quantitative PCR(qPCR)was used to investigate the CAR 10 expression in LUAD tissues and corresponding adjacent normal tissues,Immunohistochemical method(IHC)was used to detect the YB-1 and PD-L1 expression in LUAD tissues and corresponding adjacent normal tissues,The respective relationship between CAR 10,YB-1 and PD-L1 and clinicopathological characteristics and prognosis of LUAD patients was analyzed.2、TCGA was used to predict the interaction between YB-1 and clinicopathological characteristics,prognosis of LUAD patients and T cell immunity.3、qPCR was used to detect the CAR10 expression in hmnan LUAD cell lines and bronchial epithelial cells.Gefitinib-resistant LUAD subline(PC-9GR and HCC827 GR)were constructed stably.CAR10 siRNAs were used to silence CAR 10 expression in PC-9GR and HCC827 GR.Flow cytometry and CCK8 assay were used to study the effects of CAR10-depleted on cell sensibility to gefitinib.The link between CAR10 and YB-1/PD-L1 signaling pathway was detected by Western blot.CHIP assay kit were used to verify whether YB-1 acts as a transcription factor for PD-L1.4、The combined effect of CAR 10 and gefitinib was observed by Nude mice in vivo.Results:1、The expression of CAR10,YB-1 and PD-L1 in LUAD tissues was much higher than corresponding adjacent normal tissues.Increased CAR10,YB-1 and PD-L1 expression was correlated with larger tumor size,differentiation and TNM stage.2、TCGA analysis results showed that increased YB-1 expression was correlated with larger tumor size,differentiation,TNM stage and T cell immunity.3、The expression of CAR 10 was at a relatively high level in three LUAD cell lines comparing with BEAS-2B cell line.CCK8 assay showed that depletion of CAR10 could significantly inhibit the growth of PC-9GR and HCC827 GR.Flow cytometry showed that depletion of CAR 10 could increase apoptosis of PC-9GR and HCC827 GR caused by gefitinib.Colony formation assay showed CAR10 combined with gefitinib could significantly suppress the colony formation.Depletion of CAR 10 suppressed YB-1/PD-L1 signaling pathway activation,CHIP assay showed that YB-1 acted as a transcription factor for PD-L1.4、Nude mice experiments showed that CAR 10 combined gefitinib significantly inhibited tumor formation,either volume or weight.Conclusion:1、The expression of CAR 10,YB-1 and PD-L1 in LUAD tissues was much higher than corresponding adjacent normal tissues.Increased CAR10,YB-1 and PD-L1 expression was correlated with larger tumor size,differentiation and TNM stage.2、CAR 10 expression is closely related to acquired gefitinib resistance in LUAD cells.3、Silencing CAR 10 can enhance the sensitivity of gefitinib resistant cell lines to gefitinib.4、Depletion of CAR10 suppressed YB-1/PD-L1 signaling pathway activation to ameliorate gefitinib-resistance in lung adenocarcinoma celllines.