Newly Identified Mir-6770-3p Dramatically Induces Angiogenesis In Human Endothelial Cells While Uncommonly Decreasing Cell Proliferation

Micro-RNAs (miRs) are small non-coding RNAs that alter the expression of multiple mRNA targets. Although they participate in physiological processes, dysregulation of their expression is implicated in various diseases. We investigated whether miRs could be involved in the regulation of FXYD1 expression, a sub-unit of the Na+/K+-ATPase pump, that we previously described as both cardio- and vasculoprotective, under oxidative stress conditions. Using in silico analysis, we identified 3 potential miRs that could target FXYD1 mRNA: miR-3178, miR-3960 and miR-6770-3p. Using a FXYD1-3’UTR-luciferase reporter assay, we found that the overexpression of miR-6770-3p in HEK293T cells resulted in the largest reduction in luciferase activity, showing a strong targeting of FXYD1 mRNA. A mutation of the binding site in the 3’UTR of FXYD1 restored luciferase activity, confirming the binding site of miR-6770-3p. Mir-6770-3p is a novel miR that currently has no known function. MiR-6770-3p is the minor strand of its 5p/3p miR duplex in both the human endothelial cell (EC) line, EA.hy.926, and human dermal microvascular (HMVECs). Its overexpression dramatically increased endothelial tube formation in a Matrigel angiogenesis assay (>4 fold change vs. control transfection, p<0.05) and was also associated with a 2-3-fold increase of VEGFR2 and Endoglin mRNA levels, respectively, in both cell types. Conversely, it was also uncommonly associated with a decrease in cell proliferation, suggesting that the pro-angiogenic effect of miR-6770-3p was not due to increased proliferation. Interestingly and contrary to our previous findings, miR-6770-3p was the major strand in Human Umbilical Vein ECs (HUVECs). In this cell type, overexpression of the 3p strand in fact decreased tube formation, while retaining its negative effect on proliferation. The difference in miR major and minor strand may partly explain the differences in functional behaviours in HUVECs, as compared to HMVECs. In conclusion, we have identified a novel miR which has the potential to modulate vessel formation in vitro, and induce critical regulators of angiogenesis; however, further work is required to better understand how this miR performs these functions.