Hsa-miR-200c-3p is down regulated in stable coronary artery disease: A pilot study

Four male patients were enrolled for this study in collaboration with the Cardiology Unit of Policlinico Tor Vergata-Fondazione PTV (Rome). The first group of patients has chronic coronary artery disease (CAD) confirmed by coronary angiography, the second group are subjects with clinically proven healthy coronary arteries (CTR). On our case study we have performed a genome-wide methylation study on genomic DNA bisulfite-converted and a miRNA-sequencing study using NextSeq 500 ILLUMINA platform. The methylation study showed different methylated regions (DMRs) and single CpG sites (DMCs) in patients sharing the same clinical and pathological features, allowing detecting distinctly different methylation patterns between CTR subjects and CAD patients. Moreover, miRNA-sequencing results displayed a differential expression of several significant miRNAs (p-value<0.05), defining a peculiar miRNAs profile in patients featuring the same clinical data. miRNA-sequencing and genome-wide methylation integreted results, showed hsa-miR-200c-3p down-regulated in CAD patients compared to control subjects (FC CAD=2.97 and p≤0.05) and with two hypermethylated sites (genomic coordinates: chr12:7073122-7073122 and chr12:7072599-7072599) in its promoter region (p-value=0.009). We extended the validation of these results on all case study (n=96; 24 CTR and 72 CAD). Analysis of genome-wide methylation level and miRNA-seq expression profile of two different groups of patients (CTR, CAD). In total we have analyzed 4 samples.本研究与罗马托尔韦尔加塔综合医院-PTV基金会（Policlinico Tor Vergata-Fondazione PTV）心内科合作，共纳入4名男性患者。第一组为经冠状动脉造影确诊的慢性冠状动脉粥样硬化性心脏病（coronary artery disease, CAD）患者，第二组为经临床证实冠状动脉健康的对照（control, CTR）受试者。本案例研究对亚硫酸氢盐转化后的基因组DNA开展全基因组甲基化研究，并使用ILLUMINA NextSeq 500平台进行miRNA测序分析。甲基化研究结果显示，在临床与病理特征一致的患者中存在差异甲基化区域（differentially methylated regions, DMRs）与单CpG位点差异甲基化区域（differentially methylated CpG sites, DMCs），可清晰区分对照受试者与CAD患者的甲基化模式差异。此外，miRNA测序结果显示多个具有统计学意义的差异表达miRNA（p值<0.05），在临床特征一致的患者中形成独特的miRNA表达谱。整合miRNA测序与全基因组甲基化分析结果发现，与对照受试者相比，CAD患者中hsa-miR-200c-3p表达下调（CAD组折叠变化FC=2.97，p≤0.05），且其启动子区域存在两个高甲基化位点（基因组坐标：chr12:7073122-7073122与chr12:7072599-7072599，p值=0.009）。本研究将上述结果在全部案例样本（n=96，其中24名对照受试者、72名CAD患者）中进行验证。本研究分析了两组患者（CTR、CAD）的全基因组甲基化水平与miRNA测序表达谱，共纳入4份样本。