miR-20487-5p/SERCA1/MAPK/ERK Pathway Regulates Newt Limb Regeneration

Cynops orientalis, a member of the Salamandridae family, can regenerate severely injured limbs throughout its lifespan. The molecular mechanisms regulating limb regeneration in C. orientalis remain largely unknown. SERCA1 (sarcoplasmic/endoplasmic reticulum calcium ATPase 1) is a major sarco/endoplasmic reticulum calcium pump in skeletal muscle, and our previous transcriptomic sequencing results showed increased SERCA1 mRNA expression during the early stages of newt limb regeneration. In the present study, we investigated the role and regulatory mechanism of SERCA1 during newt limb regeneration by using a limb-amputation model, shRNA-mediated knockdown, Thapsigargin-mediated SERCA1 inhibition, miRNAomics analysis, dual-luciferase reporter assays and Agomir therapy. Knockdown of SERCA1 expression or suppression of SERCA1 activity impaired skeletal muscle cell dedifferentiation, disrupted AEC and blastema formation, reduced cell proliferation, and significantly delayed limb regeneration compared with control animals. Through miRNAomics analysis and in vitro dual-luciferase reporter assays, we identified miR-20487-5p as a negative regulator of SERCA1 expression in C. orientalis. In vivo treatment with miR-20487-5p Agomir down-regulated SERCA1 expression resulted in limb-regenerative defects. Mechanistically, shRNA-SERCA1 or miR-20487-5p Agomir treatment reduced ERK phosphorylation in regenerating limb tissue, suggesting an association between the miR-20487-5p/SERCA1 axis and MAPK/ERK activation. Taken together, our study supports the significant role of the miR-20487-5p/SERCA1/MAPK/ERK axis in early newt limb regeneration. This study expands the current understanding of molecular mechanisms initiating newt limb regeneration.