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Objective: We recently showed that the human long non-coding RNA, SMILR, promotes vascular smooth muscle cells (vSMCs) proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action.
Methods and Results: We used deep RNA-sequencing of human saphenous vein smooth muscle cells stimulated with IL1α and PDGF-BB with SMILR-knockdown (siRNA) or -overexpression (lentivirus), to identify SMILR regulated genes. This revealed a SMILR-dependent network essential for cell-cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ~10% increase in binucleated cells. SMILR-pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery smooth muscle cells and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 min clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR-knockdown led to a marked decrease in proliferation from ~29% in controls to ~5% with SMILR depletion.
Conclusion: Collectively, we demonstrate that SMILR is a critical mediator of vascular smooth muscle cell proliferation via direct regulation of mitotic progression. Our data further reveals a potential SMILRtargeting intervention to limit atherogenesis and adverse vascular remodelling.
- Long non-coding RNA;
- vascular remodelling
- Vascular smovoth muscle cells
- cell cycle
- blood vessels
- growth factors
- muscle cells
- noncoding RNA
- saphenous vein
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