Transcriptional changes upon overexpression of seven candidate miRNAs in human saphenous vein smooth muscle cells. [HSVSMC]

Proliferation of vascular smooth muscle cells (vSMCs) following injury is a crucial factor contributing to pathological vascular remodelling. MicroRNAs (miRNAs) are powerful gene regulators and attractive therapeutics agents. Here, we aim to systemically identify and characterise miRNAs with therapeutic potential in targeting aberrant vSMC proliferation. We performed a high-throughput in vitro screen using a library of 2042 human miRNA-mimics for their impact on VSMC proliferation and identified seven novel antiproliferative miRNAs i.e miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b.Overexpression of these 7 miRNAs affects proliferation of vSMCs from different vascular beds. Focusing on vein graft failure, a condition in which miRNA based therapeutics can be applied to the graft ex-vivo, we showed that these miRNAs reduced human saphenous vein SMC (HSVSMC) proliferation without inducing apoptosis or senescence, and five of them also significantly decreased migration.We performed RNA sequencing on HSVSMC overexpressing each of these 7 miRNAs. This analysis showed that each miRNA overexpression affects a core cell cycle gene network. However, this effect is mediated by distinct miRNA targets. Overall design: After inducing quiescence by culturing the cells in 0.2% FBS for 48h, human saphenous vein smooth muscle cells were transfected with miRNA mimics of the 7 miRNA candidates for six hours (miR-1827, miR-4774-3p, miR-5681b, miR-449b-5p, miR-491-3p, miR-323a-3p, and miR-892b or mimic Control) then cultured in the presence of IL1a and PDGF-BB for 48h to promote a pro-proliferative phenotype. Three biological replicates were performed with each replicate corresponding to cells derived from different patients. RNA sequencing was performed on HSVSMCs treated with each mimic miRNA, a control mimic or the transfection reagent only. We also performed RNA-seq on quiescent cells and cells treated with IL1a+ PDGF-BB.

血管平滑肌细胞（vascular smooth muscle cells, vSMCs）在损伤后的增殖是导致病理性血管重构的关键因素。微小核糖核酸（microRNAs, miRNAs）是一类强效的基因调控因子，亦是极具潜力的治疗试剂。本研究旨在系统性筛选并鉴定具备靶向异常vSMC增殖治疗潜力的miRNAs。我们利用包含2042个人类miRNA模拟物的文库开展高通量体外筛选，以检测其对VSMC增殖的影响，最终鉴定出7种新型抗增殖miRNAs，即miR-1827、miR-4774-3p、miR-5681b、miR-449b-5p、miR-491-3p、miR-323a-3p以及miR-892b。上述7种miRNAs的过表达可影响不同血管床来源的vSMC增殖。针对可通过离体应用miRNA疗法进行干预的静脉移植失败这一病症，我们证实这些miRNAs可抑制人大隐静脉平滑肌细胞（human saphenous vein smooth muscle cell, HSVSMC）的增殖，且不会诱导细胞凋亡或衰老；其中5种miRNAs还可显著降低细胞迁移能力。我们对过表达上述7种miRNAs的HSVSMC开展了RNA测序（RNA-seq）分析，结果显示每种miRNA的过表达均会影响核心细胞周期基因网络，但该效应通过不同的miRNA靶标介导。实验设计概述：将细胞于0.2%胎牛血清（fetal bovine serum, FBS）中培养48小时以诱导静息态后，使用7种候选miRNA模拟物（miR-1827、miR-4774-3p、miR-5681b、miR-449b-5p、miR-491-3p、miR-323a-3p、miR-892b）或阴性对照模拟物转染HSVSMC，转染时长为6小时；随后将细胞置于含IL-1α与血小板衍生生长因子BB（platelet-derived growth factor BB, PDGF-BB）的培养基中培养48小时，以诱导促增殖表型。本研究设置3次生物学重复，每一次重复均取自不同供体患者的细胞。我们对分别经各miRNA模拟物、阴性对照模拟物或仅转染试剂处理的HSVSMC开展了RNA-seq；此外，我们还对静息态细胞以及经IL-1α+PDGF-BB处理的细胞进行了RNA测序。