Effects Of MRNA,miRNAs And IncRNAs Expression On Clopidogrel Resistance In Patients With Coronary Artery Disease

Backround:Clopidogrel is an antiplatelet agent and an inactive prodrug that needs to be converted to clopidogrel active metabolite through the hepatic cytochrome P450s in order to exhibit its antiplatelet effects.Previous studies have indicted that about 15%-40%patients fail to achieve their expected antiplatelet effects when treated with standard doses of clopidogrel,which is named as clopidogrel resistance.However,the mechanism of clopidogrel resistance is not yet fully understood.Studies have shown that the expression level of ABCC3 mRNA and miR-223 in peripheral blood is related to clopidogrel resistance,miR-150 in liver tissue is related to the metabolic enzyme of clopidogrel,and miR-542-3p may be related to the metabolic enzyme of clopidogrel.Objectives:To investigate whether altered expression of some mRNA,miRNA,and lncRNA could be associated with the presence of clopidogrel resistance in clopidogrel-treated patients with coronary artery disease.Methods:PCI outpatients treated with clopidogrel were enrolled,and their clinical data and blood samples were obtained.The antiplatelet effect of clopidogrel was categorized according to the platelet aggregation profiles.Individuals with platelet aggregation values within the first quartile(Q1,?1?)were considered clopidogrel sensitive,while those who had platelet aggregation within the fourth quartile(Q4,>10?)were considered clopidogrel resistance.1.Expression of ABCC3 mRNA on clopidogrel resistance in patients with coronary artery disease:select 47 clopidogrel sensitive patients and 47 clopidogrel resistant patients,extract white blood cell total RNA,and use Agilent 2100 Bioanalyzer to carry out total RNA quality control.Quality standard were the total amount?0.11?g,RIN?6,28S/18S?0.7.The qualified specimens were used to detect ABCC3 mRNA in peripheral blood leukocytes by SYBR Green fluorescence qPCR.2.Expression of miR-223-3p on clopidogrel resistance in patients with coronary artery disease:select 47 clopidogrel sensitive patients and 47 clopidogrel resistant patients,extract white blood cell total RNA.Total RNA quality inspection was performed using Agilent 2100 Bioanalyzer.Quality standard were the total amount?0.14?g,RIN?6,28S/18S?0.7.The qualified specimens were used to detect miR-223-3p in peripheral blood leukocytes by Taqman qRT-PCR.3.Expression of miR-150 and miR-542-3p on clopidogrel resistance in patients with coronary artery disease:5 clopidogrel sensitive patients and 5 clopidogrel resistance patients were selected,and the differential expression of miRNA was screened by miRNA microarray.miR-150 and miR-542-3p are selected for qRT-PCR verification according to the results of miRNA microarray and the literature.4.Expression of lncRNAs on clopidogrel resistance in patients with coronary artery disease:5 clopidogrel sensitive patients and 5 clopidogrel resistance patients were selected,and the differential expression of lncRNA was screened by lncRNA microarray.NONHSAT083775.2,NONHSAT107804.2 and NONHSAT133455.2 are selected for qRT-PCR verification according to the results of lncRNA microarray.Results:1.Patients in the two groups had similar leukocyte ABCC3 mRNA levels using SYBR Green PCR(P=0.76).2.Patients in the two groups had similar leukocyte miR-223-3p levels using TaqMan qPCR(P=0.94).3.The miRNA microarray suggested that 21 miRNAs in peripheral leukocytes were up-regulated and 31 miRNAs down-regulated in clopidogrel resistant patients compared with clopidogrel sensitive patients(P<0.05).qRT-PCR indicated that patients in the two groups had similar plasma miR-150 and miR-542-3p levels using TaqMan qPCR(P=0.082,0.341).4.The IncRNA microarray suggested that 11 lncRNAs in peripheral leukocytes were up-regulated and 8 lncRNAs down-regulated in clopidogrel-resistant patients compared with clopidogrel-sensitive patients(P<0.05).qRT-PCR indicated that two lncRNAs(NONHSAT083775.2 and NONHSAT107804.2)in leukocytes were up-regulated and one IncRNA(NONHSAT133455.2)was down-regulated in the cases of clopidogrel resistance,consistent with the results observed by lncRNA microarray.Conclusions:1.Not only leukocyte ABCC3 mRNA and miR-223-3p,but also plasma miR-150 and miR-542-3p are not associated with altered platelet responses to clopidogrel in patients with coronary artery disease.2.Clopidogrel resistance is associated with up-regulation of lncRNA NONHSAT083775.2 and NONHSAT107804.2 and down-regulation of NONHSAT133455.2 in patients with coronary artery disease.