Estrogen Mediates CircZNF423 as a Sponge for oar-miR-541-3p to Target CALM3 for Regulating Myoblast Proliferation in Sheep
[Background]Estrogen is the main hormone secreted by female mammalian ovarian tissue,and it plays an important regulatory role in muscle growth and development.The circRNA has been found to be involved in a variety of signaling pathways related to muscle growth and development.[Objective]According to the whole transcriptome integration analysis of ovariectomized and not ovariectomized Sunite sheep in our previous study,the circZNF423 was found to regulate the expression of oar-miR-541-3p/CALM3 as ceRNA.To further investigate the molecular mechanism of estrogen in sheep muscle growth,the effect of exogenous addition of estrogen-mediated regulation of oar-miR-541-3p/CALM3 by circZNF423 on myoblast proliferation was examined by sheep primary myoblast in vitro culture.This study could provide a theoretical basis for further study of the role of estrogen and circRNA in sheep growth and development trait,and also provide new research ideas for sheep molecular design breeding.[Method]In this study,the sheep longissimus dorsi tissue was collected,and the sheep primary myoblast were isolated and cultured in vitro.The location of circZNF423 expression in myoblast was determined by immunofluorescence staining,RNA in situ hybridization(FISH)and nucleoplasmic separation experiments.RNA hybrid online software was used to predict the existence of binding relationship between circZNF423,oar-miR-541-3p and CALM3,and the binding of circZNF423 to oar-miR-541-3p,oar-miR-541-3p and CALM3 was verified by dual luciferase activity assay and biotin-labeled miRNA pull-down assay.The synthetic circZNF423 overexpression or interference plasmids,oar-miR-541-3p mimics(mimics)or inhibitors(inhibitors),and CALM3 overexpression or interference vectors were constructed in vitro and transfected in primary sheep myoblast,and the expression of proliferation markers and myoblast proliferation rate were detected using RT-qPCR,Western blot,EdU and CCK-8.To further clarify the role of estrogen in sheep muscle growth and development,the exogenous estradiol(E2)was added at different concentrations in vitro,and the changes in the expression of sheep myoblast proliferation were detected using RT-qPCR,Western blot,CCK-8,and EdU.[Result]Immunofluorescence staining showed that the isolated primary myoblast were sheep myoblast and could be used for subsequent functional validation experiments.RNA in situ hybridization and nucleoplasmic isolation experiments showed that circZNF423 was mainly expressed in the cytoplasm of sheep myoblast.The results of RNAhybrid,dual luciferase activity assay and biotin-labeled miRNA pull-down assay showed a significant binding relationship between both circZNF423 and oar-miR-541-3p,oar-miR-541-3p and CALM3 3'UTR.The expression of proliferation markers PCNA,CDK2 and Pax7 in sheep myoblast was significant increased after circZNF423/CALM3 inhibition or oar-miR-541-3p overexpression(P＜0.05 or P＜0.01).The EdU and CCK8 results indicated that the proliferation rate of sheep myoblast was significantly increased after circZNF423/CALM3 inhibition or oar-miR-541-3p overexpression(P＜0.05),while the opposite was true after circZNF423/CALM3 overexpression or oar-miR-541-3p inhibition.The expression of sheep myoblast proliferation markers was significantly higher at 10 nmol·L-1 than that at 1 nmol·L-1 and 100 nmol·L-1 after the addition of different concentrations of exogenous estradiol(E2)(P＜0.05 or P＜0.01).The mRNA and protein expression levels of the proliferation markers PCNA,CDK2 and Pax7 significantly increased in sheep myoblast,while the expression of circZNF423 and CALM3 were significantly decreased and the expression of oar-miR-541-3p was significantly increased(P＜0.05 or P＜0.01).The results of EdU and CCK8 showed that the proliferation rate of sheep myoblast was significantly increased after the addition of estradiol in vitro(P＜0.05).[Conclusion]CircZNF423 as ceRNA regulated the binding and expression of oar-miR-541-3p and CALM3 in sheep myoblast,and the addition of exogenous estrogen promoted the proliferation of sheep myoblast by inhibiting the circZNF423/oar-miR-541-3p/CALM3 pathway.These results provided a theoretical basis for revealing the molecular mechanisms of estrogen and circZNF423 in the developmental traits of sheep skeletal muscle.
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