贝母素乙对人胃癌奥沙利铂耐药细胞的影响及其机制实验研究OA

Objective:To investigate the effects of peiminine on oxaliplatin(L-OHP)-resistant human gastric cancer cells,and to explore its underlying mechanism and core target.Methods:Differentially expressed genes related to gastric cancer were screened based on The Cancer Genome Atlas stomach adenocarcinoma dataset(TCGA-STAD)and GSE65801 dataset.GO and KEGG enrichment analyses were performed to clarify pathway correlations,and core genes were obtained from the intersection of gastric cancer differentially expressed genes,autophagy-related genes and peiminine targets.Multivariate Cox regression analysis was used to analyze the prognostic impact of core genes in gastric cancer patients.SwissDock was applied to verify the molecular docking between peiminine and serine/threo-nine protein kinase 3(AKT3).In vitro cellular experiments,L-OHP-resistant human gastric cancer HGC-27/L-OHP cells were divided into five groups:control group(routine culture without treatment),L-OHP group(intervention with 20 μmol/L L-OHP),peiminine group(intervention with 50 μmol/L peiminine),L-OHP+peiminine combined group(co-intervention with 20 μmol/L L-OHP and 50 μmol/L peiminine),and L-OHP+peiminine+autophagy in-hibitor 3-methyladenine(3MA)group(co-intervention with 20 μmol/L L-OHP,50 μmol/L peiminine and 5 mmol/L 3MA).CCK-8 assay was adopted to detect cell proliferation;Transwell assay was used to evaluate cell migration and invasion abilities;DHE and JC-1 staining were performed to detect reactive oxygen species(ROS)level and mito-chondrial membrane potential;RT-qPCR was used to measure mRNA expression of key genes as well as autophagy-and apoptosis-related proteins;Western blot was utilized to detect protein expression of key genes and autophagy-and apoptosis-related proteins.Results:A total of 10 core genes were screened out,among which AKT3 served as an inde-pendent risk factor affecting the prognosis of gastric cancer patients.The binding energy of molecular docking be-tween peiminine and AKT3 was-9.158 kcal/mol.In vitro cellular experiments showed that compared with 0 μmol/L pei-minine,the OD450 values of cells treated with 25,50 and 100 μmol/L peiminine decreased sequentially at 24 h,48 h and 72 h(all P＜0.05).Compared with the control group and L-OHP group,the number of migrated and invasive HGC-27/L-OHP cells,ROS fluorescence intensity,as well as mRNA and protein expression levels of AKT3,alpha-1A adrenergic receptor(ADRA1A),kinase insert domain receptor(KDR),ubiquitin-binding protein p62 and B-cell lym-phoma-2(Bcl-2)were significantly reduced in the peiminine group and L-OHP+peiminine group;meanwhile,the proportion of JC-1 aggregates,and mRNA and protein expression levels of microtubule-associated protein light chain 3B(LC3B),PTEN-induced putative kinase 1(PINK1),Beclin1,Cleaved Caspase-3 and Bcl-2-associated X protein(Bax)were elevated sequentially(all P＜0.05).Compared with the L-OHP+peiminine group,3MA reversed all the above changes of indicators.Conclusion:Peiminine may downregulate AKT3 to promote autophagy and apoptosis ofL-OHP-resistant human gastric cancer cells,ameliorate cellular oxidative stress and mitochondrial function,and sup-press cell proliferation,migration and invasion.