Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients.
Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients.
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DOI：
10.1371/journal.pone.0181562
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Wang HW
中科院分区：
文献类型：
--
作者：
Chang TY;Tsai WC;Huang TS;Su SH;Chang CY;Ma HY;Wu CH;Yang CY;Lin CH;Huang PH;Cheng CC;Cheng SM;Wang HW
内皮细胞集落形成细胞（ecfc）是内皮祖细胞（EPCs）的一种特定细胞系，其功能损伤与冠状动脉疾病（CAD）的严重程度高度相关，冠状动脉疾病是最常见的心血管疾病（CVD）类型。越来越多的证据表明，CAD患者体液中循环的microRNAs （miRNAs）作为生物标志物具有很大的潜力。然而，我们对循环miRNA在调节ecfc功能和CAD进展中的作用的认识仍处于起步阶段。我们发现，当健康志愿者的ECFCs与条件培养基或纯化的CAD ECFCs外泌体孵育时，CAD ECFCs的分泌因子会失调健康ECFCs的迁移和成管能力。众所周知，外泌体通过引入包括mirna在内的rna来影响受体细胞的生理。通过小RNA测序（smRNA-seq），我们破译了健康个体和冠心病患者血浆循环miRNA谱，发现冠心病患者血浆miRNA谱与健康对照有显著差异。有趣的是，健康和冠心病ecfc的smRNA-seq显示，与健康对照相比，冠心病患者血浆中表达较高的12种mirna在冠心病ecfc中的表达也较高。这一结果提示这些mirna可能参与了ECFC功能的调控。为了鉴定CAD患者中差异表达的miRNA的潜在mRNA靶点，我们使用cDNA微阵列分析来鉴定CAD ecfc中下调的血管生成相关基因，并使用Pearson’s correlation来鉴定与已鉴定的血管生成相关基因负相关的miRNA。RT-qPCR分析CAD患者血浆和ECFC中与血管生成相关基因下调负相关的5个mirna，结果显示miR-146a-5p和miR-146b-5p较健康对照组上调。在CAD ecfc中敲低miR-146a-5p或miR-146b-5p可增强病变ecfc的迁移和成管活性。相反，健康ECFC中miR-146a-5p或miR-146b-5p的过表达抑制了ECFC的迁移和小管形成。TargetScan分析显示，miR-146a-5p和miR-146b-5p靶向许多在CAD ecfc中下调的血管生成相关基因。敲低miR-146a-5p或miR-146b-5p可恢复CAD ecfc中CAV1和RHOJ的水平。报告细胞实验证实miR-146a-5p和miR-146b-5p直接结合并抑制RHOJ mRNA的3 ' -UTR， RHOJ是内皮细胞血管生成电位的正调节因子。一致地，RHOJ敲低抑制ecfc的迁移和成管能力。总的来说，我们发现了CAD患者血浆和ecfc中miR-146a-5p/RHOJ和miR-146b-5p/RHOJ轴的失调，这可以作为CAD和其他血管生成相关疾病的生物标志物或治疗靶点。
Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases.