Influence Of PSMA2 Promoter SNP On Clopidogrel Response In Patients With Acute Ischemic Stroke And The Mechanism

Objective: This study aimed to confirm the influence of PSMA2 promoter single nucleotide polymorphism(SNP)rs774082 on clopidogrel response in patients with acute ischemic stroke,and to explore the mechanism of PSMA2 rs774082 polymorphism in regulating PSMA2 expression.Methods: The peripheral blood samples were collected from patients suffered from acute ischemic stroke treated with clopidogrel,and the MAR% was determined by turbidim etry after7 or more days of clopidogrel administration.CYP2C19*2,CYP2C19*3 and PSMA2 rs774082 polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP)and sanger sequencing,respectively.The effects of CYP2C19 and PSMA2 rs774082 genotypes on MAR% were analyzed.Platelets and blood cells were isolated from peripheral blood samples of patients with coronary artery disease,and RNA samples were extracted.PSMA2 m RNA expression was detected by quantitative real-time PCR,and PSMA2 rs774082 polymorphism was also genotyped.Comparisons of PSMA2 m RNA expression level among rs774082 genotypes in whole blood and the platelets were performed.Plasmids for reporter assay bearing PSMA2 promoter sequence flanking the rs774082 and rs647618 loci(two SNPs in complete linked disequilibrium)were constructed,and the effect of rs774082 and rs647618 polymorphisms on the promoter activity was investigated by dual luciferase reporter assay with or without interference of potential transcript factors.EMSA were used to explore whether rs647618 polymorphism increases the transcriptional activity of the promoter by affecting histones binding.HEK293,Hela and MEG-01 cells were treated with series concentrations of histone deacetylase inhibitor Panobinostat.The m RNA and protein expression of PSMA2 were detected by real-time q PCR and Western blotting,respectively.Wistar rats were treated with clopidogrel(intragastric administration)and Panobinostat(by intraperitoneal injection).MAR% was continuously monitored to explore the influence of Panobinostat treatment on platelet reactivity and clopidogrel response.Proteins and RNA from various tissues of the rats were extracted to explore the effect of Panobinostat on the expression of PSMA2.Results: 1)In acute IS patients,MAR% of the patients with steady state obtained after maintenance clopidogrel administration was significantly decreased as compared with baseline.The MAR% of patients carrying the PSMA2 rs774082 G allele was significantly higher than that of wild type homozygotes(rs774082 AA)patients.2)In CAD patients,PSMA2 m RNA expression in patients carrying the PSMA2rs774082 G allele was significantly increased.Dual luciferase reporter assay showed that PSMA2 promoter activity of the plasmids carrying the haplotypes bearing the PSMA2 rs647618 T allele was significantly increased.Both results from luciferase reporter assay and EMSA showed that oligonucleotides with the PSMA2 rs647618 T allele showed increased H3K27 ac binding.3)Panobinostat influenced the expression of PSMA2 in both cultured cells and in rats.In Wistar rats,Panobinostat injected intraperitoneally can lead to increased platelet reactivity.Conclusion: In acute IS patients,carriers of the PSMA2 rs774082 G allele showed decreased clopidogrel response.PSMA2 rs774082 may regulate the expression of PSMA2 at the transcriptional level by affecting H3K27 ac binding.HDAC inhibitor Panobinostat affects the expression of PSMA2 and reduced clopidogrel response.