The influence of miR-331-3p on vascular remodeling with emphasis on vascular smooth muscle cells

MicroRNAs (miRs) evolved as interesting targets to influence cellular functions and gained importance in treating atherosclerosis. Regulating VSMC proliferation through microRNA modulation presents potential therapeutic strategies to prevent atherosclerotic progression. Human Coronary Artery Smooth Muscle Cells (HCASMCs), located in the tunica media, are responsible for vascular contraction and extracellular matrix production. During atherosclerosis, LDL infiltration and inflammatory cytokine release induce HCASMC proliferation and migration into the tunica intima, where they differentiate into various phenotypes contributing to plaque formation.
This study investigates the influence of miR-331-3p on cellular functions of HCASMCs in comparison to Human Coronary Artery Endothelial Cells (HCAECs) to examine its function in atherosclerosis.

Expressions of miR-31-5p in vascular cells (HCAEC and HCASMC) as well as in murine tissue was assessed by qRT-PCR. The targeted overexpression and downregulation of miR-331-3p was achieved by pre- and anti-miR transfection. Subsequently, the effect of miR-31-5p on cellular functions such as migration, proliferation, and cell death was investigated. By subsequent in silico research possible targets of miRNA-331-3p were identified and verified on mRNA and prospective on protein levels. Immunfluorescene microscopy was performed to determine morphological changes.

Preliminary work demonstrates upregulation of miR-331-3p in vivo in ApoE-knockout mice – an atherosclerosis model – between two weeks and six months (p<0.0001). After expression studies in mouse tissues, we explored expression levels of miR-331-3p in human HCASMCs and HCAECs under different conditions like senescence, growth conditions or hypoxia. Replicative senescent HCASMCs reveal a low miR-331-3p level in vitro and in vivo, while an upregulation occurs in replicative senescent HCAECs. Cytokines like IFNγ, IL1-β and TNFα can regulate miR-331-3p level in HCASMCs, but hypoxia primarily affects HCAECs. Whereas pre-miR transfection enhanced migrational capacity, proliferation and cell viability in HCASMCs (p<0.05), anti-miRs act in reverse. Particularly, anti-miR transfection appears to have an opposing effect on functional parameters in HCAECs and HCASMCs. No morphological changes were found in HCASMCs and HCAECs after transfection with pre- or anti-miR. Cell death assay revealed anti-apoptotic tendencies of miR-331-3p. Subsequent in silico research revealed NRP2, SMAD2, TGFBR1, DUSP5, ADAM19, BRD4 and SP1 as potential targets — initial experiments confirmed their upregulation 24 and 48 hours after miR-331-3p overexpression (p<0,05). Suitably, initial data imply much less target regulation in HCAECs.

Summarizing, miR-331-3p might act as a potential key player in atherosclerosis, due to its impact on vascular remodeling. MiR-331-3p regulates functions and properties of HCASMCs less or differently than in HCAECs, which makes this miR interesting for potential therapies of cardiovascular diseases.