Cardiac sinus venosus differentiation is regulated by the expression of Hox genes under miR-23b, miR-130a and miR-106a modulation
Authors: Garcia-Martinez V , Garcia-Padilla C , Duenas A , Garcia-Lopez V , Aranega A , Franco D , Lopez-Sanchez C 
Published: June 2022

Abstract

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

Introduction
MicroRNAs have been explored in different organisms and considered molecular switches that modulate cellular differentiation and specification. Moreover, Hox gene families have gained pivotal relevance, being broadly conserved during evolution, and their roles to shape body embryo have been widely described in multiple species. Both molecular factors have shown to be essential during embryonic development, including the cardiovascular system.

Purpose
Analysis of the expression profiles of different microRNAs during early cardiac development, with special emphasis on those displaying marked expression in the sinus venosus, and correlation of these findings with several Hox family members revealed as targets of these microRNAs.

Methods
Using whole mount in situ hybridization in developing chick embryo with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a and miR-106a expressions during early stages of embryogenesis (HH3 to HH17). Those findings were correlated with putative microRNA target genes by means of mirWalk and TargetScan analyses. Through PCR analysis we explored the expression profile of all cranially expressed Hox genes, including all paralogues (Hoxa to Hoxd) from 1 to 6 in chicken cardiac sinus venosus, at stages HH11 and HH15. Finally, we developed in vitro microRNA gain-of-function experiments on cardiomyoblasts -derived from undifferentiated H9c2 cells- together with an analysis of chicken sinus venosus explants.

Results
Our results demonstrate a dynamic expression pattern from the primitive streak to cardiac looping stages for miR-23b, miR-130a and miR-106a, with marked expression in the cardiac sinus venosus. We observe that all those Hox genes analyzed -except Hoxa1- are detected at HH11, while Hoxa1, Hoxd3 and Hoxd4 are not detected at HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and sinus venosus explants, we observe the modulation of the different Hox cluster genes (Hoxa, Hoxb, Hoxc and Hoxd) by those microRNAs. Finally, we demonstrate, through a dual luciferase assay, that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a.

Conclusion
This study presents several novel findings in the field of cardiac development. Our data show a dynamic expression profile of miR-23b, miR-130a and miR-106a, and we also identify the expression of several Hox genes in the sinus venosus at stages HH11 and HH15. Moreover, we demonstrate a negative modulation of several Hox genes by these microRNAs, both in cardiomyoblasts and sinus venosus. The convergent expression of these microRNAs regulating Hox gene expressions in the sinus venosus supports the hypothesis of their roles in differentiation and compartmentalization of the cardiac venous pole.