Inhibition of RhoA and Cdc42 by miR-133a modulates retinoic acid signaling during early development of the posterior cardiac tube segment

Type of funding sources: Public Institution(s). Main funding source(s): Junta de Extremadura with FEDER co-financing.

It has been demonstrated that multiple microRNAs play crucial roles in diverse biological processes, including cardiovascular development. In particular, we have previously observed miR-133a expressing at different stages of the primitive streak precardiac cells until the formation of the cardiac tube. MiR-133a role has also been reported as a modulator of proliferation and differentiation during heart development, involving many target genes and signaling pathways. Furthermore, we have shown in previous studies that retinoic acid plays a crucial role in cardiac development, modulating the expression of atrial markers.

The aim of this work is to analyze the miR-133a modulation role on the retinoic acid signaling pathway in the posterior segment of the heart tube during cardiac looping formation.

By means of microinjections in both primitive endocardial tubes of chick embryo culture, gain- and loss-of-function experiments were performed with premiR-133a and anti-miR-133a, respectively. Subsequently, embryos were subjected to whole mount in situ hybridization with Tbx5 and AMHC1 probes, and qPCR. After TargetScan analysis and luciferase assays, immunohistochemistry was carried out by using anti-RhoA, -Cdc42 and -Raldh2/ALDH1A2 antibodies.

Luciferase assays show that miR-133a is capable to recognize and bind to RhoA and Cdc42 3´UTR, thus triggering their mRNA degradation. Our results reveal that miR-133a gain-of-function experiments repress the expression of atrial markers Tbx5 and AMHC1, as well as RhoA, Cdc42 and -Raldh2/ALDH1A2 signals. Supporting these data, miR-133a loss-of-function experiments show the opposite effect: they increase the expression of Tbx5 and AMHC1, as well as RhoA, Cdc42 and Raldh2/ALDH1A2 signals.

Our results suggest that miR-133a could modulate negatively retinoic acid signaling by triggering degradation of pivotal genes RhoA and Cdc42, both required for Raldh2/ALDH1A2 expression, crucial to the synthesis of retinoic acid.