The involvement of microRNAs in the pathogenesis of systemic lupus erythematosus-related vascular calcification

Tandel, Shikha Mahesh (2018) The involvement of microRNAs in the pathogenesis of systemic lupus erythematosus-related vascular calcification. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Background Patients with Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease, have an elevated risk of developing cardiovascular disease compared to their healthy counterparts. The chronic inflammatory state in these patients heighten their risk of developing premature atherosclerosis and subsequent progression to coronary artery calcification (CAC). CAC increases the risk of coronary events occurring and it is characterised by the osteogenic transdifferentiation of coronary artery smooth muscle cells (CASMCs) and consequent deposition of hydroxyapatite minerals within the extracellular matrix. Runx2 is a potent regulator of osteogenic differentiation of CASMCs and its expression is regulated by microRNAs (miRNA or miR). CAC is a major clinical problem, but limited treatment options are available. This study investigates whether miRNA alteration affects mineralisation in an in vitro calcification assay model. Aims This study aimed to validate the association of miR-3148 with Runx2-driven CAC and investigate the effects of different miRNAs on genes involved in CAC in CASMCs. Methods Human Embryonic Kidney (HEK) 293T cells were used to produce high-titre lentiviral vectors for miR-3148 and its sponge, and pLL3.7 plasmid. Human Coronary Artery Smooth Muscle Cells (CASMCs) were transfected with lentivirus and cultured in osteogenic media. The level of miRNA-3148 in cells and microvesicles derived from TNF-α stimulated CASMCs in vitro was determined. Alizarin Red S staining and quantification was used to identify changes in mineralisation between the different groups. Gene expression analysis was conducted. Results miR-3148 levels were higher in TNF-α stimulated CASMCs compared to control. Stable transfection was observed in lentivirus transfected cells. A lower level of calcium deposits was observed in cells transfected with miR-3148 lentivirus in comparison to empty vector control (P = 0.00201) and transfection with miRNA-3148 sponge. miRNA-3148 also lowered Runx2-gene expression in relation to control and miR-3148 sponge at day 4. Conclusion The current study demonstrates that overexpression of miR-3148 reduces the level of mineralisation via Runx2 in vitro, suggesting a protective role for miRNA-3148 against osteogenic transdifferentiation of CASMCs. These data imply the possibility of using microvesicles and/or miRNAs for drug therapy in the management or treatment of CAC.