| Myoz3 gene is one of the members of myozs family and an important component of muscle Z-line.It plays an important role in maintaining the structure and function of skeletal muscle.However,we do not know its effect on myocyte proliferation and apoptosis and its transcriptional regulation mechanism.Therefore,in this study,we first detected the mRNA expression pattern of Myoz3 gene,and explored the effect of Myoz3 on the proliferation and apoptosis of chicken skeletal muscle satellite cells by qPCR,CCK-8,edu,flow cytometry and other technologies.Then,we cloned the 5 ’transcriptional regulatory region of Myoz3 gene by PCR,and analyzed the structure and function of Myoz3 promoter by bioinformatics.The transcriptional regulation mechanism of MyoD on Myoz3 gene was studied by site directed mutation,overexpression and interfering transcription factors,EMSA and ChIP method.The main results of this study are as follows:(1)Ralative mRNA expression of Myoz3 gene in breast muscle was significantly higher than that in leg muscle.There were obvious differences in the expression of Myoz3 during E15-D35,which were as follows: the expression of E15-D6 gradually increased,peaked during D2-D6,and decreased sharply at D27 and D35.In satellite cells cultured in vitro,the mRNA expression of Myoz3 gene increased first and then decreased.(2)After overexpression / interference with Myoz3 gene,the relative expression of PCNA,CDK2 and Cyclin D1 in skeletal muscle satellite cells increased / decreased significantly(P < 0.05).(3)The results of CCK-8 and EdU showed that after overexpression and interference with Myoz3 gene,the cell viability of chicken satellite cells and the proportion of EdU positive proliferating cells were significantly increased and decreased(P < 0.05).At the same time,flow cytometry showed that the increase of Myoz3 expression made more satellite cells stay in G2 and S phase,while the decrease of Myoz3 expression made more satellite cells stay in G1 phase.(4)The expression of Bcl-2 and caspases-3 decreased significantly(P<0.05),while the expression of p53 increased significantly(P<0.05),but the expression of apoptosis related genes did not change significantly(P>0.05)after overexpression of Myoz3.Interestingly,we found that the overexpression and interference of Myoz3 did not significantly change the number of apoptosis by flow cytometry.(5)The core promoter of Myoz3 gene was identified in the range of-152 / + 240 by double luciferase reporter gene test,and two binding site(E-box)of transcription factors MyoD was predicted here.Site-directed mutation test showed that the mutation of MyoD binding site significantly reduced the promoter transcription activity.Overexpression of MyoD significantly enhanced the transcriptional activity of the promoter.(6)Overexpression of transcription factor MyoD significantly increased the expression level of Myoz3 in satellite cells(P<0.05),while the interference of MyoD significantly decreased the expression level of Myoz3 in satellite cells(P<0.05).At the same time,the mRNA expression trends of myoz3 and MyoD were consistent in different stages of growth and development.The results of EMSA and ChIP assay showed that the transcription factor MyoD was bound to the chicken Myoz3 core promoter sequence in vitro and in vivo.In conclusion,the expression of myoz3 gene is closely related to the growth and development of chicken muscle before and after birth,and is positively regulating the proliferation of skeletal muscle satellite cells,and the transcription factor MyoD is also involved in this biological process by activating the transcription of myoz3 gene. |
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