| The growth and development of skeletal muscle is a decisive factor in the quality of animal meat production,and its regulation mechanism is a highly coordinated biological process under the influence of many factors.In recent years,the study of skeletal muscle growth and development has been regarded as the breakthrough point of animal breeding improvement in animal husbandry.However,previous studies mainly focused on the regulation of animal muscle growth and development at the level of m RNA,miRNA,and Lnc RNA.There are few reports on the regulation of circRNA on muscle development.Therefore,the study of circRNA regulating bovine muscle development and its mechanism can provide a new idea for understanding the process of bovine muscle development and molecular breeding.In this study,the transcriptome sequencing was performed on muscle samples of gene-edited Luxi cattle MSTN-/-and wild type Luxi cattle MSTN-/-samples obtained in the same growth background.Myostatin（MSTN）is mainly expressed in the skeletal muscle of animals and has a negative regulatory effect on skeletal muscle growth.It can inhibit muscle growth and development by inhibiting the proliferation and differentiation of skeletal muscle cells.Knock-down of the MSTN gene will promote the growth and trait changes of the experimental group of cattle.Screening the differentially expressed circRNA on the basis of the difference in muscle growth status by transcriptome sequencing technology,so as to study whether the differences are due to the same regulatory effect on muscle growth or even the unique regulatory mechanism.A new circRNA with specific temporal and spatial expression was identified by reverse splicing of exon 1 and 2 of bovine FAM120A gene,and was named BOS-circFAM120A.In this study,bovine skeletal muscle satellite cells were used as the main materials to analyze the regulatory effects and mechanisms of circFAM120A on proliferation and differentiation of skeletal muscle satellite cells,and the main results are as follows:（1）A total of 259 circRNAs with significantly different expressions were screened through transcriptome analysis of the circRNA data detected in each test sample.5 circRNAs with different expression trends were randomly selected,and primers with ring structure characteristics were designed according to the sequence 150bp before and after the reverse binding site sequence displayed in the sequence information.The expression levels of circRNAs in the cell model interfering with MSTN were consistent with the results reported in the sequencing,which proved the reliability of the circRNA data.（2）The full-length 330nt bovine circFAM120A sequence was obtained,and the exonuclease digestion experiment showed that it had strong exonuclease resistance.The transcription inhibition experiment with actinomycin D showed that circFAM120A was formed after the precursor RNA and had the characteristics of acting as circRNA,further confirming that circFAM120A was a circRNA.（3）The expression level of circFAM120A increased gradually during the proliferation and differentiation of bovine skeletal muscle satellite cells from the proliferative stage（DM）to the post-induced differentiation stage（DM1 DM2 DM3）,and the highest expression level was found on the third day of differentiation.The tissue expression profile showed that circFAM120A was widely expressed in all tissues of fetal cattle at 3,6,9 months of age and adult cattle.Among the muscle expressions,the expression of longissimus dorsi muscle was significantly higher than that of other tissues in adult cattle.In the MSTN-knockdown model,the expression of circFAM120A decreased during the proliferation phase,increased significantly during the differentiation phase,and increased with the degree of cell differentiation.These results indicated that circFAM120A had a reverse regulatory effect on the proliferation and differentiation of bovine skeletal muscle satellite cells.（4）The expression of markers related to skeletal muscle proliferation was promoted and the expression of markers related to skeletal muscle differentiation was inhibited after circFAM120A knock-down.Under the microscope,the cell density of bovine skeletal muscle satellite cells after circFAM120A knock-down was significantly higher than that of the control group at the proliferation stage,and the diameter and differentiation status of muscle canal at the differentiation stage were lower than those of the control group.The results of CCK8 cell proliferation assay indicated that interference with circFAM120A promoted the differentiation of bovine skeletal muscle satellite.Flow cytometry results further demonstrated that interference with circFAM120A inhibited the differentiation of bovine skeletal muscle satellite cells.（5）Overexpression of circFAM120A promoted the expression of skeletal muscle differentiation-related markers and inhibited the expression of skeletal muscle proliferation-related markers.The results of microobservation,CCK8,and flow cytometry experiment further showed that circFAM120A significantly promoted differentiation and inhibited proliferation of bovine skeletal muscle cells.（6）Bioinformatics analysis predicted that bovine circFAM120A had binding sites with five miRNAs containing miR-128.The results of subcellular localization showed that most of circFAM120A were located in the cytoplasm,suggesting that circFAM120A may have the function of sponge-absorbing miRNA.The expression level of circFAM120A was detected by overexpression of five miRNAs respectively,and the results showed that circFAM120A might interact with miR-128/miR-378.After overexpression of circFAM120A,the expression of miR-128 showed a opposite trend compared with the control group.,suggesting an interaction between circFAM120A and miR-128.（7）The dual luciferase reporter gene system verified that miR-128 could bind to circFAM120A.（8）The results of time-sequential expression of miR-128 in bovine skeletal muscle satellite cells showed that miR-128 gradually increased with the cell growth process.The miR-128mimics were transfected into bovine skeletal muscle satellite cells,and the overexpression of miR-128 was detected by CCK8 assay.The results showed that miR-128 inhibited the proliferation of bovine skeletal muscle satellite cells and the expression of skeletal muscle proliferation related markers,while promoted the expression of skeletal muscle differentiation related markers compared with the control group.These results indicated that miR-128 could inhibit proliferation and promote differentiation in bovine skeletal muscle satellite cells.It was further suggested that circFAM120A played a role by regulating the expression of target genes by adsorption of miR-128.（9）Bioinformatics analysis was performed to analyze the target genes binding to miR-128,and the expression of MSTN was significantly decreased after the overexpression of miR-128.After overexpression of circFA120A,the expression of MSTN was significantly up-regulated,while knockdown was down-regulated.The dual luciferase reporter gene system comfirmed that miR-128 could bind to MSTN.There was no significant change in the expression level of circFAM120A after overexpression of MSTN,which further indicated that there was no direct interaction between circFAM120A and MSTN.The circFAM120A plays a role in skeletal muscle growth and development by regulating the expression of its target gene MSTN throufh miR-128 as a bridge,while MSTN promotes the production of circFAM120A through the inhibitory effect of myostatin.In conclusion,the results suggest that circFAM120A regulates the proliferation and differentiation of bovine skeletal muscle satellite cells by targeting the absorption of miR-128 to reduce the inhibition of MSTN.In this study,we identified a novel circRNA（BOS-circFAM120A）that regulates the growth process in bovine muscle,and predicted and verified the regulatory mechanism of circFAM120A’s c RNA network that inhibates proliferation by competitively binding with miR-128 to block MSTN expression.The results of this study enrich the functional studies of the involvement of circRNAs and have important significance for further elucidating the molecular mechanism of animal muscle cell differentiation. |
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