| Background:The morbidity and mortality of lung cancer ranks first among malignant tumors worldwide.Small cell lung cancer(SCLC),accounting for about 20%of lung cancer,is the most aggressive subtype of lung cancer,and nearly 90%of patients have a survival period of less than 5 years.Most SCLC patients have tumor invasion of surrounding tissues and lymphatic or distant metastasis at the early stage of diagnosis.Therefore,chemotherapy(cisplatin combined with etoposide)is the main approach in clinical treatment for SCLC.The initial response rate is high,but secondary chemotherapy resistance is very likely to cause recurrence.Chemotherapy resistance and the poor survival rate of SCLC patients has become an important issue which was urgently needed to be solved in clinical treatment and scientific research.Our research group previously showed that cell autophagy is closely related to chemotherapy resistance in SCLC,but its potential molecular mechanism has not been completely clarified due to the complexity of the biomolecular regulatory network.Previous studies have shown that nuclear receptor-binding factor 2(NRBF2)could participate in the initial phase of autophagy and regulate autophagy process,but the antitumor effect of NRBF2 and the mechanism of NRBF2 regulating autophagy have not been fully elucidated.Purpose:In this study,we plan to further explore the mechanism of NRBF2-mediated drug resistance and the role of autophagy in this process.This research could further enrich the study of drug resistance mechanisms in SCLC.The study of drug resistance-related target genes provides a theoretical basis for the treatment of chemotherapy resistance in SCLC.Experimental design:(1)Analyze NRBF2 gene expression differences between chemotherapeutic resistant and sensitive cells of SCLC by immunoblotting,and construct corresponding NRBF2 down-regulated and over-expressed lentivirus stable transgenic cells.(2)The effects of NRBF2 on chemotherapy resistance were analyzed in vitro.At the same time,naked animal models were applied to verify whether NRBF2 affected chemotherapy resistance in vivo.(3)The relationship between NBRF2 and cell autophagy was studied by transmission electron microscope(observation of cell autophagosomes),laser confocal microscope(observation of autophagy double fluorescence)and immunoblot(detection of autophagy marker proteins expression).(4)DNA Pull Down,chromatin immunoprecipitation assay(ChIP),and luciferase reporter gene system were applied to screen and verify upstream transcription factors that regulated NRBF2 gene.Co-immunoprecipitation and liquid chromatography-tandem mass spectrometry(LC-MS-MS)were applied to study the mechanism of NRBF2-induced autophagy.(5)We applied RNA sequencing to identify the molecular signal pathways regulated by NRBF2,and then we investigated whether existing targeted drugs such as tyrosine kinase inhibitors could affect chemotherapy resistance in SCLC.Result:(1)NRBF2 could promote chemotherapy resistance in SCLC by regulating autophagy.(2)NRBF2 protein directly interacted with P62 protein,which promoted the formation of P62 bodies and enhanced autophagy.(3)The transcription factor XRCC6 promoted the expression of NRBF2 by directly binding to the promoter of the NRBF2 gene.(4)EPHA2 was involved in NRBF2-induced drug resistance process of SCLC.The EPHA2-related tyrosine kinase inhibitor sitravatinib could reduce the chemotherapy resistance in SCLC.Conclusions:NRBF2 interacted with P62 protein to enhance autophagy,resulting in chemotherapy resistance of SCLC.In addition,EPHA2-related signal pathway was involved in NRBF2-induced drug resistance in SCLC. |
