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. 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.