Molecular Mechanism By Which CPT1C Promotes Cisplatin Resistance In NSCLC By Regulating Epithelial Mesenchymal Transition(EMT)via Snail

Background & Objective:lung cancer has become the most common tumor diagnosed by cancer worldwide,approximately 85% of patients are histological subtype of non-small cell lung cancer(NSCLC),and cisplatin is the first-line drug for the treatment of NSCLC,but NSCLC will develop resistance to the drug,which reduces the therapeutic efficacy.Therefore,it is crucial to investigate the key targets that lead to cisplatin resistance.Carnitine palmitoyltransferase 1C(CPT1C)is a fatty acid that can mediate the transport of long-chain fatty acids across the mitochondrial bilayer membrane and ultimately through the β-Oxidation,a key enzyme providing energy,is also currently attracting much attention in the field of tumor research because of its specific expression in brain tissue versus tumor tissue.So,this study mainly explores the role that CPT1 C plays in the process of cisplatin resistance and investigates its possibility as a key target for reversing cisplatin resistance.Methods:(1)The tissue expression differences of CPT1 C were obtained by TCGA database analysis,then validated by Western blot experiments,and the Kaplan Meier plotter website was used to analyze the association between CPT1 C and patient prognosis;MTT assay was used to detect the sensitivity to cisplatin in selected cells,and the effect of CPT1 C on cell viability.(2)The effects of overexpression and knockdown of CPT1 C on cisplatin sensitivity were analyzed using MTT assay,annexinv FITC / PI apoptosis detection assay,and apoptosis Hoechst staining assay assays.(3)Dual luciferase reporter assay was performed to detect the effect of cisplatin on CPT1 C promoter;The effect of cisplatin on the half-life of CPT1 C protein was examined by Western blot;Sites were matched for possible E3 ubiquitin ligases by protein structure analysis,followed by coimmunoprecipitation experiments to test whether CPT1 C interacts with E3 enzymes and the effect of cisplatin on CPT1 C ubiquitination;Finally western blot assay was used to detect the protein expression of overexpressed and knocked down NEDD4 L.(4)Scratch assay was performed to detect the effect of cisplatin on A549 or H1299 cell migration;The effects of cisplatin on EMT related proteins were detected by Western blot;Dual luciferase reporter gene experiments were performed to detect the effect of cisplatin on the Snail promoter;Bioinformatic analysis techniques probed the association of CPT1 C with EMT related proteins.(5)The effects of overexpression versus knockdown of CPT1 C on EMT related proteins were examined by Western blot;Scratch assay was performed to detect the effect of CPT1 C expression on cell migration;Reactive oxygen species detection experiments showed the effect of expression of CPT1 C on cellular ROS;Scratch assay to verify whether CPT1 C induced EMT is associated with ROS production;Western blot assay was performed to verify whether CPT1 C promoted EMT through ROS / AKT/ Snail pathway.(6)Western blot assay verified whether cisplatin promoted EMT through CPT1C;Dual luciferase reporter assay verified whether cisplatin promoted Snail promoter activity via CPT1C;Scratch assay to verify whether cisplatin induced EMT is associated with ROS generation;Reactive oxygen species detection experiments showed whether cisplatin promotes ROS generation via CPT1C;Western blot assay was performed to verify whether cisplatin promotes EMT through CPT1 Cdependent ROS / AKT/ Snail pathway;MTT assay was performed to verify whether CPT1 Cexerts cisplatin resistance dependent on ROS / AKT pathway.Results:(1)In non-small cell carcinoma cells A549,H1299,CPT1 C was found to be highly expressed compared with normal lung epithelial cells,and the differences were statistically significant(P < 0.05).In survival analysis,the probability of survival in the high expression CPT1 C group was significantly lower than that in the low expression group,and the difference was statistically significant(P < 0.05).(2)The results of MTT assay,annexinv FITC / PI apoptosis detection assay,and apoptosis Hoechst staining assay all showed that overexpression of CPT1 C could improve the cell viability when cells were treated with cisplatin compared with drug alone,and knockdown of CPT1 C could decrease the cell viability when cells were treated with cisplatin,and the differences were statistically significant(P < 0.05).(3)Cisplatin has no effect on CPT1 C promoter activity but rather reduces its ubiquitination and thereby its degradation rate,and the results of IP experiments suggest that NEDD4 L may act as an E3 ubiquitin ligase for the specific ubiquitination of CPT1C;The difference was statistically significant(P < 0.05).(4)The scratch assay results showed that cisplatin promoted A549 and H1299 cell migration;The experimental results of Western blot assay indicated that cisplatin promoted EMT related protein expression;The dual luciferase reporter assay results illustrated that cisplatin promoted the Snail promoter property;The results of the raw signal analysis indicated a positive association between CPT1 C and EMT related proteins;The difference was statistically significant(P < 0.05).(5)The results of Western blot experiments indicated a positive association between CPT1 Cand EMT related proteins;The scratch assay results indicated that the expression of CPT1 C promoted cell migration;Reactive oxygen species detection experiments showed that expression of CPT1 C promoted cellular ROS production;The scratch assay results indicated that CPT1 C induced EMT was associated with ROS production;The results of Western blot assay indicated that CPT1 C was responsible for promoting EMT through ROS / AKT/ Snail pathway;The difference was statistically significant(P < 0.05).(6)The results of Western blot experiments indicated that cisplatin was promoting EMT through CPT1C;Dual luciferase reporter assay results indicated that cisplatin promoted Snail promoter activity via CPT1C;The scratch assay results indicated that cisplatin induced EMT was dependent on ROS generation;The results of the reactive oxygen species detection experiment indicated that cisplatin promoted ROS generation through CPT1C;The results of Western blot assay indicated that cisplatin promoted EMT through CPT1 C dependent ROS / AKT/ Snail pathway;The MTT assay results indicated whether CPT1 C exerts cisplatin resistance dependent on the ROS / AKT/ Snail pathway.Conclusion:(1)In non-small cell lung cancer A549 and H1299 cell lines,CPT1 C expression was higher than that in normal lung epithelial HBE cells,and high expression of CPT1 Cwas associated with poor prognosis.(2)Overexpression of CPT1 C increased cisplatin resistance in NSCLC A549,H1299 cell lines,while knockdown of CPT1 C decreased cisplatin resistance in NSCLC A549,H1299 cell lines,thus CPT1 C is a key target for cisplatin resistance in NSCLC.(3)Cisplatin regulates CPT1 C expression at the post transcriptional level via NEDD4 L,and CPT1 C promotes EMT progression through the ROS / AKT/ Snail pathway,thereby exerting cisplatin resistance.