Evidence for a role of microRNA-21 and microRNA-125b in negatively regulating angiogenic processes

Recently discovered, miRNAs have quickly become strong regulators of biological processes. These small non-coding RNAs of about 22 nucleotides partially base pair to the 3’UTR of the targeted mRNAs and repress them. Due to their wide range effects, microRNAs were extensively studied in various diseases and were rapidly demonstrated to be deregulated
in pathologies such as cancer. More recently, they have been shown to be implicated in
vascular network formation (angiogenesis) and were proposed to be used in anti-angiogenic
therapies. Nowadays about twenty angiomiRs have been discovered including the endothelial
specific miR-126. As observed in several miRNA profiling of endothelial cells and confirmed
in our laboratory in HUVECs (human umbilical veins endothelial cells), miR-21 and miR-
125b are highly expressed in this cell type suggesting that these miRNAs could play a role in
vascular network formation. We then studied the implication of miR-21 and miR-125b in in
vitro as well as in vivo angiogenesis.
One of the most studied miRNA in cancer progression is miR-21 as it was shown to modify proliferating properties of numerous tumor cells. Our experiments revealed that miR-21 overexpression and inhibition have no direct effect on endothelial cells proliferation rate.
However, miR-21 overexpression leads to the inhibition of HUVECs migration and tube formation as demonstrated in in vitro angiogenic assays. Moreover, opposite effects were
observed upon miR-21 inhibition. We also confirmed that RhoB, a small Rho-GTPase implicated in stress fibers formation, is involved in these phenomena as RhoB inhibition using siRNA mimics miR-21 overexpression in endothelial cells. Moreover, miR-21 modulation affects RhoB mRNA and protein expressions. We further demonstrated a direct interaction between miR-21 and the RhoB 3’UTR confirming that miR-21 modulates angiogenesis partially through its effect on RhoB expression.
A similar approach was used to study the implication of miR-125b in vascular network formation. In vivo, miR-125b expression was modulated in the zebrafish revealing that miR-125b expression needs to be controlled for proper intersomitic blood vessels establishment. In
vitro, miR-125b overexpression decreases HUVECs migration and tube formation whereas
miR-125b inhibition increases these functions. A transcriptomic analysis suggests that numerous adhesion molecules such as VE-cadherin or MCAM are involved in these processes.
Furthermore, other proteins known to regulate angiogenesis such as the transcription factor
ETS1 and the VEGFA receptor, VEGFR2 were also shown to be regulated by miR-125b. This
observation confirms that miR-125b modulates angiogenic properties of endothelial cells.
Finally, we investigated the impact of miR-21 and miR-125b overexpression in an in vivo pathological model of angiogenesis. In a mouse model of choroïdal neovascularization we
demonstrated that miR-21 or miR-125b overexpression in the eyes of these mice decreases
blood vessel establishment suggesting that these microRNAs could be used as therapeutic antiangiogenic agents.
Taken together, the results presented in this thesis show that miR-21 and miR-125b regulate angiogenesis in vitro and in vivo.