Study On The Tumor Suppressive Role Of Protease-Activated Receptor-4 In Esophagealsquamous Cell Carcinoma Cells

Objective:Esophageal squamous cell carcinoma(ESCC)is 90% of esophageal cancer,and a few are esophageal adenocarcinomas.Vitamins and trace elements such as the lack of nutrients,contact with and mycin carcinogenic nitrosamines,HPV(Human Papillomavirus,HPV)and epstein-barr virus(EBV Epstein-Barr virus get,)and hepatitis B virus,hepatitis B virus comes,HBV)such as virus infection and bad living habits such as alcohol,tobacco,and other environmental factors are cause esophageal cancer,Studies have shown that PAR4 expressed in esophageal squamous cell may be due to gene promoter methylation of a 10-degree patch PAR4,prompt PAR4 gene methylation in the promoter region of the expressed in esophageal cancer have a certain influence to its,but in the role of esophageal cancer occurs is unclear,need further research.This study applies PAR4 specificity agonists PAR4 activation peptide(PAR4-AP)ESCC,incubation application spectrum of protein markers(Tandem Mass Tag,TMT)method for PAR4 activated and ESCC genes differentially expressed proteomics analysis,application of protein immunoblot(western blot),immunohistochemistry,xCELLigence DP system and cell function analyzer determined by MTT test the result and find PAR4 activation in ESCC potential therapeutic value.Methord:1.The protein changes of the squamous cell(EC9706)of the esophageal cancer(EC9706)were detected by using the Tandem Mass Tag(TMT)method.2.PAR4-AP incubation of esophageal squamous cell(EC9706),zero hour,1 hour,2 hours,6 hours,12 hours,24 hours group,extraction of total protein,Western bolt TMT method validation are the obvious change of 5 kinds of protein CHMP1 B,PURA,PARG,HIST1H2 AH and CASPASE.3.Immunohistochemical methods were used to observe the expression of PAR4,P53,PURA,PARG,CASPASE9 and HIST1H2 AH in the epithelial tissue of esophageal carcinoma.4.PAR4-AP incubation of esophageal squamous cell(EC9706),zero hour,1 hour,2 hours,6 hours,12 hours,24 hours group,extraction of total protein,Western bolt TMT method validation are the obvious change of 5 kinds of protein CHMP1 B,PURA,PARG,HIST1H2 AH and CASPASE.5.Using the xCELLigence cell function analyzer DP system,the control group was detected,and the squamous cell(EC9706)of the esophageal carcinoma(EC9706)was incubated by PAR4-AP(real-time monitoring for 72 hours)cell proliferation.6.Determined by MTT method is used to inspect PAR4-AP incubation of esophageal squamous cell(EC9706),zero hour,1 hour,2 hours,6 hours,12 hours,24 hours a day,calculate the survival rate of esophageal squamous cell.Result:1.TMT results showed that there were 5,225 proteins in esophageal cancer squamous cell.Thirty-three of these proteins changed,including 15 expressions of up-regulated protein and 18 down-regulated proteins.GO(Gene ontology)and KEGG(said 49-year-old kyoko Encyclopedia of Genes and Genomes)analysis showed that the increase of Genes including tumor apoptosis related Gene(for example CASPASE9),cut the Genes including resistance,autophagy,and promote cell proliferation and apoptosis related Gene(such as CHMP1 B,PURA,PARG and HIST1H2AH).2.Western blot detection PAR4-AP esophageal squamous cell incubation,zero hour,1 hour,2 hours,6 hours,12 hours,24 hours a day,CHMP1 B,PURA,PARG and HIST1H2 AH expression in esophageal squamous cells decreased,CASPASE9 express rise,consistent with the results of TMT.3.The expression of PAR4,p53,CHMP1 B,PURA,PARG and HIST1H2 AH in the epithelial tissue of esophageal carcinoma and normal esophageal carcinoma.(1)in immunohistochemistry,the expression of PAR4 in the epithelial tissue of esophageal cancer was decreased,and the difference was statistically significant(P < 0.05)compared with normal esophageal epithelial tissue.(2)in immunohistochemistry,the expression of variant P53 in the epithelial tissue of esophageal carcinoma was increased,and the difference was statistically significant(P < 0.05)compared with normal esophageal epithelial tissue.(3)in immunohistochemistry,the expression of CHMP1 B,PURA,PARG and HIST1H2 AH was observed in the epithelial tissue of esophageal cancer,and the difference was statistically significant(P < 0.05)compared with normal esophageal epithelial tissue.4.by xCELLigence cells analyzer DP system function,the control,PAR4-AP incubation of esophageal squamous cell(72 hours)real-time monitoring of cell proliferation,results show PAR4-AP can suppress esophageal squamous cell proliferation.5.Determined by MTT method is used to inspect PAR4-AP esophageal squamous cell incubation,zero hour,1 hour,2 hours,6 hours,12 hours,24 hours a day,PAR4-AP after incubation of esophageal squamous cell vigor significantly lower than the control group,difference has statistical significance(P < 0.01).Conclusion:1.PAR4-AP can change the protein expression profile of esophageal carcinoma squamous cell.2.the activated PAR4 may be involved in the regulation of squamous cell of esophageal cancer through apoptosis,autophagy and tumor suppressor genes.3.the activated PAR4 may inhibit the proliferation of esophageal squamous cells.