The Role Of MiR-183/96/182 Cluster In The Development Of Bovine Myoblast Cells And The Genetic Diversity Of Its Target Gene Foxo1

Muscle development is an important part of meat quality and meat production,so it has always been the focus of cattle genetic improvement research.However,muscle development involves the regulation of many regulatory factors and signaling pathways.The molecular regulation network is still not clear.There are still many relevant regulatory factors that have not been discovered.As a transcriptional regulator,FOXO1 gene is involved in the processes of muscle growth and differentiation,apoptosis,glucose metabolism,cell cycle arrest,and tumor suppression.It has a wide range of biological functions and is an important candidate for studying muscle development.In recent years,it has also been reported that miRNA plays an important role in regulating muscle development.Therefore,this study,arounding the bovine FOXO1 gene,investigated the role of miR-183/96/182 cluster in differentiation and proliferation of bovine muscle development using Dual luciferase reporter system,q RT-PCR,Western Blot,cellular immunofluorescence,CCK-8 and EdU.And using "Once PCR method" investigated the Indels within the bovine FOXO1 gene.It may provide a theoretical basis for the genetic improvement of beef cattle.Our main results are as follows:1.miR-183/96/182 cluster regulates differentiation and proliferation of bovine muscle cell by targeting bovine FOXO1 gene.After software prediction and dual luciferase report analysis,the miR-183/96/182 cluster was demonstrated to target the bovine FOXO1 gene.The results of q RT-PCR and Western Blot showed that miR-183/96/182 cluster could inhibit the expression of FOXO1 by targeting FOXO1 gene,which significantly up-regulated the expression of Myf5,Myo D and Myo G,explaining that miR-183/96/182 cluster could promote myoblast differentiation.This cluster also significantly up-regulated the expression of CDK2,Cyclin D1 and PCNA,while significantly down-regulated the expression of p21,explaining that it could promote cell proliferation.The results of cellular immunofluorescence assay explained that miR-183/96/182 cluster could promote myotube production,further verifying its fuction of myocyte differentiation.The results of CCK-8 and EdU assay explained that the miR-183/96/182 cluster could promote myoblast proliferation,further verifying its fuction of myoblast proliferation.2.The Indels within bovine FOXO1 gene could be used as a molecular marker for genetic improvement.In five Chinese cattle populations(Pinan,Xianan,Nanyang,Qinchuan and Qaidam cattle),two Indels within FOXO1 gene were screened,named Indel-3(rs383545622)and Indel-4(rs525318770).A "Once PCR method" was successfully applied.This method could improve research efficiency and reduce research costs.Population genetic analysis showed that this five Chinese cattle populations have species specificities.Association analysis showed that the Indels within bovine FOXO1 gene could be used as molecular marker for genetic improvement work.