The Regulatory Effects And Mechanism Of Akirin Gene In Duck Myoblast Proliferation And Differentiation

Akirin gene plays an important role in myogenesis.Normally,Akirin gene family contains two members,Akirin1 and Akirin2.However,only one gene(Akirin2)was identified in the birds.In order to verify the function of duck Akirin2 gene,two cell lines which was overexpressed Akirin2 or ectopic expressed Akirin1 were used as model,the regulatory effects and mechanism of Akirin2 gene on the myoblast proliferation and differentiation was determined by using RT-PCR,immunofluorescence,Western Blot and flow cytometry.The main results and conclusions are as follows:(1)The multiple alignments between birds’ genome and mouse Akirin gene showed that same paralogous gene was identified.This paralogous gene shared over 80% homology with mouse Akirin2,but only shared 41%-45% homology with mouse Akirin1.Phylogenetic tree analysis result showed that bird paralogous gene and other Akirin2 genes were clustered into one group.Functional domain analysis showed that bird paralogous gene shared a similar structure with other Akirin2 genes.(2)Duck myoblast differentiation was successfully induced.Duck Akirin2 eukaryotic vector was successfully constructed and transfected into the duck myoblast.Overexpression of Akirin2 did not significantly influence the expression of MRFs and MEF2 s,the cell cycle arrest and myotube formation.Thus,overexpression of duck Akirin2 gene cannot influence myoblast differentiation.(3)After overexpression of Akirin2 gene,the cell proliferation activity was significantly up-regulated.Although the cell cycle regulator factors(Cycline D family)had no change after overexpression of Akirin2,the expression of mTOR/S6 K signaling pathway related factors significantly increased.However,after rapamycin treatment,the positive effect of Akirin2 on myoblast proliferation was aborted.Thus,duck Akirin2 gene can regulate myoblast proliferation through mTOR/S6 K signaling pathway.(4)It was verified by dual-luciferase reporter assay system that miR-365 could bind to Akirin2 3’-UTR.Overexpression of miR-365 can significantly inhibit the expression of mTOR and S6 K.Overexpression of miR-365 can also significantly inhibit the duck myoblast activity,suggested that when the expression of Akirin2 was inhibited by miR-365,the expression of mTOR and S6 K was down-regulated,and then the cell proliferation was influenced.Thus,these results further supported that duck Akirin2 gene can regulate myoblast proliferation through mTOR/S6 K signaling pathway.(5)Ectopic expression of Akirin1 can significantly increase the expression of MRF4,and MEF2 B.Moreover,the expression of p38 MAPK and PI3K/Akt signaling pathway related factors were significantly up-regulated.After adding the SB203580,the expression of p-p38α and MRF4 significantly decreased,suggested Akirin1 gene may regulate myoblast differentiation through p38 MAPK signaling pathway.In addition,after adding the LY294002,the expression of p-Akt and MRF4 significantly decreased,suggested Akirin1 gene may also regulate myoblast differentiation through PI3K/Akt signaling pathway.In conclusion,only Akirin2 orthologous gene was found in the birds’ genome which we mentioned.Duck Akirin2 gene cannot regulate myoblast differentiation,but can regulate the proliferation.Duck Akirin2 can promote myoblast proliferation the via activation of the mTOR/S6 K signaling pathway,In addition,ectopic expression of Akirin1 can promote myoblast differentiation via p38 MAPK and PI3K/Akt signaling pathway.This study clarified the function of Akirin2 in the myoblast proliferation,and provided a foundation for further study.