Construction Of MiRNA Prognostic Model For Predicting Gastric Cancer And Research In R-flurbiprofen Promoting Apoptosis

Background: It has been reported 951,000 cases of gastric cancer with 723,000 deaths newly occured in 2012.Epidemiological Status of Gastric Cancer indicated Chinese accounted for 42.6% and 45.0% of the world's new gastric cancer morbidity and mortality in 2017.The 5-year overall survival rate of advanced gastric cancer was less than 30%.Combining multiple related microRNAs can improve the accuracy of prognosis,which has broad practical prospects and provides the possibility of individualized medical treatment.Previous studies have shown that R-flurbiprofen has anti-tumor effects.There are no research on flurbiprofen or R-flurbiprofen with microRNA in tumor cases now.Therefore,it is important to detect R-flurbiprofen's regulation of microRNAs on tumor's growth,apoptosis,migration and invasion.Objective:Initially,enantioselective esterification of(R,S)-flurbiprofen catalyzed by Candida sp.Lipase was conducted in ionic liquid.Explore the optimum reaction condition,purified R-flurbiprofen.Secondly,to identify an miRNA model set with prognostic power for patients with gastric adenocar-cinoma.Finally,to verify R-flurbiprofen's regulation on 5miRNAs and the promoting mechanism on apoptosis in GC cells.Method and result:Under optimal conditions(flurbiprofen(0.2 mmol),methanol(2 mmol),CSL(4 mg/mL),[BMIM][PF6](5 mL),50° C),CSL exhibited a satisfying enzyme performance(average enzyme activity,261.5 ? mol/g/h;E value,20.3).The enzymatic esterification can be scaled up easily.Furthermore,CSL exhibited only a slight decrease in catalytic performance after six repetitions.Purification had been done by using SHIMADZU-8A high performance liquid chromatography,preparative chromatographic column chiralpak AD-H.miRNA-seq data from 155 patients and 37 controls were downloaded from The Cancer Genome Atlas(TCGA)database for a comprehensive analysis of miRNA expression profiles and were used as training data.A total of 5 prognostic miRNAs,which have not been previously reported,were identified using univariate and multivariate Cox regression analyses.A separate 155-patient TCGA cohort was used as a validation set for evaluation of the risk model.Patients in the training set were assigned into high-and low-risk groups according to the 5-miRNA signature risk scores.Kaplan-Meier survival analyses demonstrated that patients with high risk scores had significantly shorter survival times than those with low risk scores.The risk model validation confirmed the prognostic ability of this 5-miRNA signature in predicting the risk status of patients.Stratification analysis for clinical prognostic variables demonstrated recurrence and age were significant prognostic factors in the low-and high-risk groups,respectively.In conclusion,the present 5-miRNA signature is a potential independent risk factor for patient outcomes.The risk model based on the 5-miRNA signature performed well in predicting overall survival time in patients with gastric.After 48 hr treatment of 400 ?M R-flurbiprofen,the proliferation of MGC-803 gastric cancer cells was significantly weakened compared with the control group.IL-1? and TNF-a in the supernatant of the cell were significantly increased compared with the control group.Real-time PCR results showed that miRNA-3687,miRNA-548 o,KBTBD11 and CAMK1 D decreased significantly;microRNA-7-2,microRNA-9-3,microRNA-1255 a,CXCL5,ADPGK,ATM,TRIM66,Bax,Caspase 3,Vimentin and N-Cadherin increased significantly,KLF12,FRRS1,Bcl-2 and E-Cadherin was not regulated significantly.Western blot results showed that Bax and Vimentin increased significantly,while Caspase 3 did not differ;cell invasion experiment showed that cell invasion and migration increased,but there was no significant difference compared with the control group.The apoptosis increased significantly compared with the control group.Conclusion:We disclosed an efficient lipase-catalyzed enantioselective esterification of(R,S)-flurbiprofen in IL.The anion and alkyl chain of the imidazolium cation greatly influenced the catalytic performance of lipase.Under the optimal conditions,CSL exhibited a satisfying catalytic performance in [BMIM][PF6].The scale-up esterification of(R,S)-flurbiprofen was also examined,and a high enantiomeric excess for(S)-flurbiprofen(ee > 99%)was achieved when the conversion was up to 62% in 30 h.The present 5-miRNA signature is a potential independent risk factor for patient outcomes.The risk model based on the 5-miRNA signature performed well in predicting overall survival time in patients with gastric.In the experimental group,5-miRNAs and their target genes was regulated significantly.Up-regulated the expression of Bax gene and protein,inhibited the proliferation of gastric cancer cells and promoted their apoptosis.5-miRNA may regulate tumor cell immunity,cytokine cytokine receptor pathway by raising IL-6,IL-1?,TNF-?,CXCL5.Up regulating of Bax,Caspase3 promote GC cell apoptosis.Up regulation of ADPGK suggested that target aerobic metabolism can increase tumor cell sensitivity to drugs.Down regulation of Mir-548 o induces increasing in CXCL5.Correspondence of miRNA548 o and target gene CXCL5 may confirmed by double fluorescein enzyme reporting gene.By CXCL5/miRNA knockout,The invasion/metastasis mechanism of gastric cancer may be further studied in the experiment of nude rat tumor.