Transcriptional changes upon overexpression of seven candidate miRNAs in human saphenous vein endothelial cells. [HSVEC]

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.In contrast to HSVSMC, miRNA overexpression on saphenous vein endothelial cells (ECs) led to no or lower decrease of proliferation for the seven miRNAs. We performed RNA-seq on HSVEC overexpressing the seven miRNAs and showed a limited response of ECs to the miRNA overexpression. After inducing quiescence by culturing the cells in 0.2% FBS for 12h, human saphenous vein endothelial 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 10% FBS for 48h to promote a 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 untransfected cells.

血管平滑肌细胞（vascular smooth muscle cells，vSMCs）在损伤后的增殖是引发病理性血管重构的关键诱因。微小RNA（microRNAs，miRNAs）是一类强效的基因调控因子，同时也是极具开发前景的治疗制剂。本研究旨在系统性地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实验，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。聚焦于静脉移植失败这一可通过离体移植给药的miRNA治疗适用病症，我们证实这些miRNAs可抑制人隐静脉平滑肌细胞（human saphenous vein SMC，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。与HSVSMC不同，对隐静脉内皮细胞（endothelial cells，ECs）过表达上述7种miRNAs后，其增殖仅出现轻微或无明显下降。
我们对过表达7种miRNAs的人隐静脉内皮细胞（human saphenous vein endothelial cells，HSVEC）开展RNA测序（RNA-seq），结果显示内皮细胞对miRNA过表达的应答反应有限。在使用0.2%胎牛血清（fetal bovine serum，FBS）培养细胞12小时以诱导细胞静息后，我们将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）或对照模拟物转染至人隐静脉内皮细胞，随后将细胞置于10% FBS中培养48小时以诱导增殖表型。本实验设置3次生物学重复，每一次重复均取自不同供体患者的细胞。我们还对分别转染各miRNA模拟物、对照模拟物以及未转染细胞的HSVSMCs开展了RNA测序。