摘要： 旨在探究bta-miR-133a参与肉牛调控背最长肌发育的分子机制。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，数据筛选与肉牛骨骼肌发育相关的bta-miR-133a，采用生物信息学分析软件鉴定其保守性并预测其靶基因，对其靶基因进行GO富集分析和KEGG通路富集分析，并通过双荧光素酶报告试验对预测的潜在靶基因进行验证；选取C2C12细胞进行功能验证，过表达和敲除bta-miR-133a，48 h后2%马血清诱导观察肌管分化过程，CCK-8检测细胞增殖率，实时荧光定量PCR检测细胞增殖分化标记基因PCNA、CCND1、Myh1、Myod1以及靶基因的表达量。结果表明，bta-miR-133a的成熟序列在各物种间高度保守，靶基因预测为PAX7 ，经双荧光素酶报告试验验证，bta-miR-133a与PAX7存在靶标关系。GO富集和KEGG通路分析结果显示，预测靶基因显著富集于肌动蛋白细胞骨架组织的调节、经典Wnt信号通路的负调控、肌动蛋白丝结合等GO条目中和MAPK、Ras、FoxO等与肌肉发育相关的信号通路中。过表达bta-miR-133a促进肌管分化（P <0.01），在72 h，降低细胞的增殖率（P <0.01），抑制靶基因PAX7的表达（P <0.01）；敲除bta-miR-133a则抑制肌管分化（P <0.01），在72 h时，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，过表达bta-miR-133a降低其标记基因PCNA、CCND1的表达量（P <0.05），敲除组则相反；在细胞分化方面，过表达bta-miR-133a增加其标记基因Myh1、Myod1的表达量（P <0.01），敲除组则相反。综上表明，bta-miR-133a可能通过靶向负调控PAX7的表达抑制成肌细胞增殖、促进其分化而参与背最长肌的发育过程。

关键词: 布莱凯特黑牛, 鲁西黄牛, bta-miR-133a, PAX7, C2C12细胞

Abstract: The aims to explore the molecular mechanism of bta-miR-133a involved in the regulation of longissimus dorsi development in beef cattle. The longissimus dorsi muscle of Black cattle and Luxi cattle was collected for sRNA database construction, and the data was used to screen bta-miR-133a related to the development of beef cattle skeletal muscle. Use bioinformatics analysis software to identify its conservation and predict its target genes, perform GO enrichment analysis and KEGG pathway enrichment analysis for its target genes, and verify the predicted potential target genes through the dual luciferase report test. C2C12 cells were selected for functional verification, bta-miR-133a was overexpressed and knockouted. After 48 h, 2% horse serum was used to observe the myotube differentiation process, CCK-8 to detect cell proliferation rate, Real-time quantitative polymerase chain reaction to detect the expression of cell proliferation and differentiation marker genes PCNA, CCND1, Myh1, Myod1 and target genes. The results showed that the mature sequence of bta-miR-133a was highly conserved among various species. The target gene was predicted to be PAX7. The dual luciferase report test verifies that bta-miR-133a had a target relationship with PAX7.The result of GO enrichment and KEGG pathways analysis showed that the predicted target genes were significantly enriched in the regulation of actin cytoskeleton organization tissue, the negative regulation of the classical Wnt signaling pathway, actin filament binding and other GO components, and MAPK, Ras, FoxO and other signaling pathways related to muscle development in. Overexpression of bta-miR-133a promoted myotube differentiation(P <0.01), at 72 h, reduced cell proliferation rate(P <0.01), inhibited the expression of target gene PAX7 (P <0.01);knocked out bta-miR-133a inhibited myotube differentiation(P <0.01), increased cell proliferation rate at 72 h(P <0.05), and promoted the expression of target gene PAX7 (P <0.01). In terms of cell proliferation, overexpression of bta-miR-133a reduced the expression level of its marker genes PCNA and CCND1 (P <0.05), while the knockout group was the opposite. In terms of cell differentiation, overexpression of bta-miR-133a increased the expression level of the marker genes Myh1 and Myod1 (P <0.01), while the knockout group was the opposite. In summary, bta-miR-133a may participate in the development of longissimus dorsi by targeting and negatively regulating the expression of PAX7 to inhibit the proliferation of myoblasts and promote their differentiation.

Key words: Black cattle, Luxi cattle, bta-miR-133a, PAX7, C2C12 cell

中图分类号: 

• S813.1