| Salamander is an ideal model for studying limb regeneration.After amputation,dedifferentiation of cells in various tissues at the stump site proliferates and migrates to the vicinity of the stump together with fibroblasts in the surrounding connective tissue to form the blastema,which cells re-differentiate to develop a complete limb.Among them,the proliferation and migration of mesenchymal cells and fibroblasts are the basis for the formation of blastema,which is essential for limb regeneration.Coronins are known to play a role in cell movement and migration,but what role Coronins play in the process of limb regeneration and how it affects the process of limb regeneration is unclear.In this study,we first constructed a newt amputated limb regeneration and defect model and found that Coronin 1A was up-regulated after salamander amputation.The transcription level of Coronin 1A(Coro1A)was analyzed by real-time quantitative reverse transcription PCR and in situ hybridization.The protein level of Coronin1 A in limb regeneration was analyzed by western blotting and immunofluorescence staining,it was found that the expression of Coronin 1A first increased and then decreased.It reached a peak at 7～14 days after amputation,then began to decline,and returned to normal levels at 42 days after amputation.Using the previous sequencing results of the research group and miRanda analysis,the miRNAs that bind to the 3’-UTRs of Coronin 1A were screened and further verified by realtime quantitative PCR and dual-luciferase reporter.Coronin1 A is targeted by miR-27b-3p.Treatment of amputated salamanders with the miR-27b-3p agonist Agomir-27 b showed that Coronin1 A protein expression was down-regulated in limb stump sites in vivo,and the process of limb regeneration was inhibited.This study helps to understand the role and molecular mechanism of Coronin 1A in the process of limb regeneration,and provides a theoretical basis for exploring the cellular activities and molecular regulation mechanism of amputated limb regeneration in salamanders. |
