Analysis of microRNAs miR-20a-3p and miR-203a-3p in the regulation of cellular dedifferentiation in the zebrafish model

Abstract

Tissue regeneration is a biological process evolutionarily conserved in large vertebrate groups. In teleosts it is characterized by the stages of dedifferentiation, reorganization and differentiation of cells adjacent to injured or amputated tissue, giving rise to a new structure, identical morphologically and functionally to that lost. Advances in understanding the biological mechanisms underlying regeneration have been achieved by studying the caudal fin in the zebrafish (Danio rerio), a teleost species with remarkable capability to regenerate damaged tissues. At the cellular level, this process occurs primarily from the dedifferentiation of mature osteoblasts in osteoprogenitor cells, followed by adequate repopulation of the bone parts that have been lost in the lesion. At the molecular level, it involves the activation and fine-tuning of gene expression, coordinated by the interaction between transcription factors and non-coding RNAs, respectively. The sp7 gene, a key transcription factor for bone differentiation and also a marker for mature osteoblasts, exhibits high levels of expression in the region adjacent to the caudal fin lesion, which progressively decrease distally to the amputation site, suggesting that it exerts important role in the de-differentiation of osteoblasts in zebrafish. Similarly, several microRNAs (miRNAs) - small regulatory endogenous non-coding RNAs - have been associated with regeneration in zebrafish, although their exact functional role in cell dedifferentiation needs to be elucidated. Bioinformatic prediction analyzes identified microRNAs miR-20a-3p and miR-203a-3p as potential post-transcriptional regulators of sp7 activity, which was reinforced by previous studies that reported the expression of both in bone tissues during regeneration, especially in the dedifferentiation of osteoblasts. This work aims to test whether the candidate miRNAs (miR-20a-3p and miR-203a-3p) physically interact and modulate the activity of the sp7 transcription factor associated with the control of dedifferentiation in the zebrafish model in vivo. The validation of this interaction will allow mapping the functional role of microRNAs and enriching the regulatory pathways associated with this primordial step of the regeneration process. It is also intended to test if this potential target miRNA interaction is evolutionarily conserved, by means of extended bioinformatic prediction to species of large groups of living vertebrates. The knowledge generated regarding the functionality of specific microRNAs as mediators of dedifferentiation, the essential cellular mechanism of the regeneration process, may be applied in the future to manipulate regulatory pathways that allow controlled alterations in the differentiation state of the cells in the lesion region. This subproject is an integral part of current research (Regular Project FAPESP process 2018 / 05484-0) which aims to characterize the functional role of microRNAs during the regeneration process.