Circulating MicroRNAs As Biomarker For Antiplatelet Drug Resistance

Backgroud:With the development of society,coronary heart disease has been the one of important diseases which harm human health.Intervetion therapy based on stent implatation is the leading method for coronary heart disease.Thus,dual anti-platelet therapy which combine aspirin and clopidogrel has become the cornerstone.However,even though dual anti-platelet therapy has been achieved completely,some study showed that the side effect happened in 3-30% patients who took oral clopidogrel.Considered that clopidogrel resistance caused in-stent restenosi,the adverse cardiovascular events happened.There are lots of ways for monitoring clopidogel resistance,such as LTA,VASP,VerifyNow and TEG,but few are adapted in the clinical practice.microRNAs(miRNAs)are a class of endogenous and 18-20 bp length noncoding small RNA.miRNAs can take effect through binding target gene and regulate gene expression at the post-transcription level.Recently,people have focused on the studies of miRNAs as the novel biomarker for kinds of diseases.Objective: Make sure micro RNAs which can be as the biomarker for antiplatelet drug resistance in the circulation so that we can adjust the drug therapy plan according to the micro RNAs.Methods: The patients who were diagnosed in-stent restenosis through coronary angiography were inrolled.These patients matched with inclusion criteria and exclsion criteria.These patients should take oral asipirin 300 mg and clopidogrel 300 mg for 24 hours or 600 mg 6 hours or asipirin 100 mg and clopidogrel 75mg/d for 7days before coronary angiography,then we drawed A?B?C three bottles of blood which included2ml?2ml and 6ml from the antecubital vein,and A and B bottles were used for VerifyNow detective device in order to give PRU value.Then we divided these patients into two groups,the patients whose PRU value?208 went to the low-responce resistance group and the other went to normal-responce group.Otherwise we choosed randomly 1 samples from every group to perform microarray assay so as to primarily screen differetially expressive miRNAs.Combining the previous paper we choosed candidates of miRNAs.From the samples of the above two groups,we extracted total RNA and measured the contcenration and purity.After that,we choosed synthetic C.elegans miR-39 as endogenous control and we used qRT-PCR to verify the candidate miRNAs betweenthe two groups,then differentially expressive miRNAs were conformed.According to the data,we analysed the correlation between miRNAs and clopidogrel resistance.Otherwise,to the patients of low responce group,we changed the drug plan(double dose of clopidogrel and ticagrelor),then some patients had no low responce.Considering that,we gave another three groups:group A was low responce group(n=4)before double dose of clopidogrel was adapted and normal responce group(n=4)after double dose of clopidogrel was adapted,group B was low responce group(n=3)before double dose of clopidogrel was adapted and low responce group(n=3)after double dose of clopidogrel was adapted,group C was low responce group(n=13)before ticagrelar was taken and normal responce group(n=13)after ticagrelar was taken.We verfy the micro RNAs from the plasma of the above three groups by qRT-PCR.Results:(1)According to the PRU value,the patients were divdied into two groups including low-respongce group(n=20)and normal-responce group(n=27).Comparative the two groups' baseline,we know that age?RBC and HBG were significant different btween the two groups,however,there is no significant difference about BMI,proportion of hypertetion and diabetes mellitus,WBC and PLT,liver and renal function,the length and diameter of stent.Logistic analysis showed that age ? RBC and HBG would not influence clopidogrel resistance.Mciroarray assay showed that there were 76 miRNAs which has 2 fold change between the two groups,refering to previous paper we choosed12 miRNAs including miR-16?miR-223?miR-191?miR-155?miR-146?miR-26a?miR-26 b ? miR-24 ? miR-150 ? miR-126-5p ? miR-126-3p ? miR-23 a.(2)Through qRT-PCR,there were 4 significantly different miRNAs including miR-223?miR-191?miR-155 ? miR-146.The result showed that comprared with normal-responce group miR-223 and miR-191 were significantly increased,however,miR-155 and miR-146 were significantly decreased(P<0.05).Combination miR-223?miR-191?miR-155?miR-146 can increase the dianosis potency of clopidogel resistanc to 92.2% and the correlation with PRU value was negative(r=0.645,P<0.001).After we adjusted the antiplatelet drug,the result of group A showed that miR-146 expression level in low responce group was significantly decreased than that in normal responce group(P=0.004),however,there was no significant different in group B(P>0.05).In group C,miR-155 expression level of low responce group was significantly decreased(P=0.002)and miR-146 expression level of low responce group was significantly increased(P=0.004)than that of normal responce group.Conclusion: expression of miR-223 and miR-191 increased,miR-155 and miR-146 expression decreased,combination of changes of the four miRNAs can be used to diagnose clopidogrel resistanc and the diagnosis potency was high.They can be used as potential biomarker for antiplatelet drug resistanc.