Department of Biotechnology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal 576104, Karnataka, India.
Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal 576104, Karnataka, India.
Life Sci. 2020 Mar 15;245:117364. doi: 10.1016/j.lfs.2020.117364. Epub 2020 Jan 28.
To investigate the impact of microRNA target SNPs (mirSNPs) and their interaction with miRNAs on important drug-metabolizing enzymes, transporters and target genes for prediction of clopidogrel drug response in cardiovascular disease individuals.
A prospective cross-sectional study was conducted on 292 individuals undergoing clopidogrel drug therapy. All the enrolled participants were administered 300 mg loading dose followed by 75 mg dose of maintenance therapy. Platelet aggregations were measured before administration of the loading dose and 2 h post fifth day dose of clopidogrel maintenance therapy. Clopidogrel carboxylic acid metabolite from plasma and urine were analyzed post maintenance therapy using the RP-HPLC method. Genotyping of mirSNP's shortlisted through in silico analysis was performed by tetra ARMS PCR and validated by Sanger DNA sequencing. The levels of selected miRNAs were estimated by the TaqMan-PCR assay. Functional validation of mirSNPs was performed in HepG2 cells after transfecting with the selected gene and miRNA mimics. Protein expressions were analyzed by western blot.
23% of enrolled individuals showed resistance to clopidogrel therapy. Out of 13 mirSNP's analyzed, CYP2C19 rs4244285 was associated with clopidogrel drug resistance and clopidogrel carboxylic acid metabolite in urine and plasma. hsa-miR-1343-3p and hsa-miR-6783-3p levels were significantly high in individuals with CYP2C19 rs4244285 mutant genotype and these miRNAs down-regulated the protein expression of CYP2C19.
We demonstrated the role of coding mirSNP (rs4244285) in the regulation of the CYP2C19 gene through miRNAs and its implications to clopidogrel drug response prediction in the Indian population.
研究微小 RNA 靶标单核苷酸多态性（mirSNP）及其与微小 RNA 的相互作用对重要药物代谢酶、转运体和靶基因的影响，以预测心血管疾病患者氯吡格雷药物反应。
对 292 例接受氯吡格雷药物治疗的个体进行了前瞻性横断面研究。所有入组患者均给予 300mg 负荷剂量，然后给予 75mg 维持剂量。在给予负荷剂量前和氯吡格雷维持治疗第 5 天第 2 小时测量血小板聚集。使用反相高效液相色谱法（RP-HPLC）分析维持治疗后血浆和尿液中的氯吡格雷羧酸代谢物。通过四面体扩增阻碍突变系统（tetra ARMS）PCR 对通过计算机分析筛选的 mirSNP 进行基因分型，并通过 Sanger DNA 测序进行验证。通过 TaqMan-PCR 检测选定微小 RNA 的水平。在转染选定基因和微小 RNA 模拟物后，在 HepG2 细胞中对 mirSNP 进行功能验证。通过 Western blot 分析蛋白表达。
23%的入组个体对氯吡格雷治疗表现出耐药性。在分析的 13 个 mirSNP 中，CYP2C19 rs4244285 与氯吡格雷药物耐药性以及尿液和血浆中的氯吡格雷羧酸代谢物相关。CYP2C19 rs4244285 突变基因型个体中 hsa-miR-1343-3p 和 hsa-miR-6783-3p 水平显著升高，这些微小 RNA 下调 CYP2C19 蛋白表达。
我们证明了编码 mirSNP（rs4244285）在通过微小 RNA 调节 CYP2C19 基因中的作用及其对印度人群氯吡格雷药物反应预测的意义。
