The Relationship Between CircFGFR2 With MiR-200a-3p And Its Role In Poultry Skeletal Muscle Cell

Circular RNA is a class of endogenous RNA with special ring structure,the diverse functions of circular RNA were acting as miRNA spong,participating in post-transcriptional control,coding protein in vitro and deriving pseudogenes.The research of circular RNA of human,mouse,monkey and pig were reported,but the kind and function of circular RNA of poultry skeletal muscle remain unknown.In this study,we aim to identify a circular RNA of poultry-circFGFR2,explore its relationship with miR-200a-3p and its potential role in poultry skeletal muscle development.According to Divergent primer PCR and RNase R exonuclease testing,we demonstrate that circFGFR2 is a well-expressed and stable circular RNA in chicken skeletal muscle.It was 636 bp in length and cyclized by 3-6 exons of FGFR2.We constructed a luciferase reporter(psi-CHECK-miR-200a-3p)to monitor miR-200a-3p activity by inserting a perfect miR-200a-3p target into the 3'-untranslated region(UTR)of Renilla luciferase.Luciferase reporter assay showed that the activity of Renilla luciferase was significantly increased when co-transfected the luciferase reporter with miR-200a-3p and circFGFR2 expression vector compared with the luciferase reporter,miR-200a-3p and empty circular RNA expression vector co-transfected group.It demonstrated that circFGFR2 could combine with miR-200a-3p and inhibit its activity to combine with the luciferase reporter,then leading to increase the activity of Renilla luciferase.Furthermore,we performed flow cytometry analysis and Ed U(5-Ethynyl-2'-deoxyuridine)assay to explore the effect of circFGFR2 on poultry skeletal muscle development,results suggested that overexpression of circFGFR2 could significantly promote QM-7 cell and chicken primary myoblast proliferation while silence of circFGFR2 could significantly inhibit QM-7 cell and chicken primary myoblast proliferation,which demonstrated that circFGFR2 play important role in poultry skeletal muscle development.In conclusion,we confirmed circFGFR2 was a well-expressed and stable circular RNA in poultry skeletal muscle.C ircFGFR2 could combine with miR-200a-3p and inhibit its activity.The role of circFGFR2 in poultry skeletal muscle development was that it could promote skeletal muscle cell proliferation.