Overexpression of microRNA126 for Vascular Repair in Atherosclerosis

MicroRNAs (miRNAs) are an emerging class of highly conserved, non-coding small RNAs that post-transcriptionally regulate gene expression by degradation of mRNA.  Recent studies have identified miR-126 as an essential mediator of vascular integrity and angiogenesis by regulating the responses of endothelial cells to vascular endothelial growth factor (VEGF). Furthermore, in atherosclerosis related pathology, it has been shown to reduce the macrophage and apoptotic cell content of plaques through increased CXCL12 expression, which has also been show to promote endothelial progenitor cell migration to repair the vasculature. microRNA-126 therefore represents a unique therapeutic potential in vascular biology and atherosclerosis.

Objective: Construct a lentivirus vector expressing miR-126 to test the hypothesis that genetically manipulated macrophage expression of miR-126 will facilitate vascular protection.

Methods: The ability of lenti-SMP-mir126 to successful transfer miR-126 into the cell and affect intracellular signaling was evaluated by infection of HEK 293T and macrophage RAW 264.7 cell line, followed by quantitative PCR.  Western blot analysis was performed to evaluate changes in CXCL12 protein expression of RAW 264.7 infected cells.  Cytotoxic effects of over-expressing miR-126 in both macrophages and HEK 293T cells were determined by MTT proliferation assay.  Lastly, the effect of Lenti-miR delivery of miR-126 to endothelial cells (EOMA) on wound healing/migration was evaluated.

Results:  Infection of macrophage RAW cell line displayed the ability of Lenti-SMP-miR126 to effectively repress its endogenous targets and increase CXCL12 protein expression. There were no significant cytotoxic effects of miR-126 on either HEK 293T or Raw 264.7 cells. Additionally, delivery of mir-126 to EOMA cells resulted in increased cell migration/wound healing.

Conclusion: We are able to successfully express mir-126 which could functionally bind and repress its downstream targets in vitro. This vector was able to alter cell migration/wound healing of EOMA cells strengthening its potential use as an atherosclerosis therapeutic.