中国畜牧杂志

2021, v.57(09) 57-64

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bta-miR-1通过调控PAX7参与骨骼肌发育的功能研究
bta-miR-1 Participates in the Functional Study of Skeletal Muscle Development Through Regulation of PAX7

于堃;刘瑞莉;刘贤勋;柏学进;董雅娟;
YU Kun;LIU Ruili;LIU Xianxun;BAI Xuejin;DONG Yajuan;College of Animal Science and Technology, Qingdao Agricultural University;Shandong Black Cattle Breeding Engineering Technological Research Center;Shandong Black Cattle Science Technology Co.,Ltd.;

摘要(Abstract):

本研究旨在探究牛miR-1(bta-miR-1)参与调控肉牛骨骼肌发育的分子机制。通过采集布莱凯特黑牛和鲁西黄牛的背最长肌进行sRNA建库,筛选与肉牛骨骼肌发育相关的bta-miR-1,采用生物信息学方法鉴定其保守性和预测其靶基因PAX7;为验证bta-miR-1的功能:在成肌细胞中转染bta-miR-1模拟剂/阴性对照(bta-miR-1 mimic/NC)和bta-miR-1抑制剂/阴性对照(bta-miR-1 inhibitor/NC),48 h后用2%马血清诱导观察肌管分化过程,CCK-8检测细胞增殖率,qRT-PCR检测细胞增殖分化标记基因PCNA、CDK2、Myog、Myod1以及靶基因PAX7的表达量。结果表明,转染bta-miR-1mimic促进肌管分化,降低细胞的增殖率(P<0.01),抑制靶基因PAX7的表达(P<0.01),转染bta-miR-1 inhibitor则抑制肌管分化,增加细胞的增殖率(P<0.05),促进靶基因PAX7表达(P<0.01);在细胞增殖方面,转染bta-miR-1 mimic降低其标记基因PCNA、CDK2的表达量(P<0.01),抑制组则相反;在细胞分化方面,转染bta-miR-1 mimic增加其标记基因Myog、Myod1的表达量(P<0.01),抑制组则相反。综上,bta-miR-1可能通过负调控靶基因PAX7促进成肌细胞分化、抑制成肌细胞增殖而参与骨骼肌的发育过程。
This study aims to explore the molecular mechanism of bta-miR-1 in the regulation of skeletal muscle development in beef cattle. By collecting the longissimus dorsi muscle of Black Cattle and Luxi Cattle for sRNA database construction, data screening of bta-miR-1 related to beef cattle skeletal muscle development, using bioinformatics methods to identify its conservation and predict its target genes PAX7. To verify the function of bta-miR-1, transfect bta-miR-1 mimic/NC and btamiR-1 inhibitor/NC into myoblasts, and observe the differentiation process of myotube with 2% horse serum after 48 h, and CCK-8 to detect cell proliferation The expression of the cell proliferation and differentiation marker genes PCNA, CDK2, Myog, Myod1 and the target gene PAX7 were detected by qRT-PCR. The results showed that transfection of bta-miR-1 mimic promoted myotube differentiation, reduced cell proliferation rate(P<0.01), and inhibited the expression of target gene PAX7(P<0.01), and transfection of bta-miR-1 inhibitor inhibited muscle Tube differentiation, increase cell proliferation rate(P<0.05), promote the expression of target gene PAX7(P<0.01); in terms of cell proliferation, transfection of bta-miR-1 mimic reduces the expression of its marker genes PCNA and CDK2(P<0.01), the inhibition group is opposite; in terms of cell differentiation, transfection of bta-miR-1 mimic increases the expression of its marker genes Myog and Myod1(P<0.01), the inhibition group is opposite. In conclusion, bta-miR-1 may participate in the development of skeletal muscle by negatively regulating the target gene PAX7 to promote myoblast differentiation and inhibit myoblast proliferation.

关键词(KeyWords): bta-miR-1;布莱凯特黑牛;鲁西黄牛;PAX7;C2C12细胞
bta-miR-1;Black Cattle;Luxi Cattle;PAX7;C2C12 Cell

Abstract:

Keywords:

基金项目(Foundation): 山东省现代化农业产业技术体系牛产业创新团队(6682216030)

作者(Author): 于堃;刘瑞莉;刘贤勋;柏学进;董雅娟;
YU Kun;LIU Ruili;LIU Xianxun;BAI Xuejin;DONG Yajuan;College of Animal Science and Technology, Qingdao Agricultural University;Shandong Black Cattle Breeding Engineering Technological Research Center;Shandong Black Cattle Science Technology Co.,Ltd.;

Email:

DOI: 10.19556/j.0258-7033.20201208-02

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