背景:运动功能的发挥取决于各种组织,如骨骼和肌肉。miR-196在癌症等领域得到了广泛的研究,但其对骨骼和骨骼肌的影响鲜有报道。为了探讨miR-196家族在骨骼和骨骼肌中的作用,我们使用先前成功构建的miR-196a-1和miR-196b基因敲除斑马鱼动物模型进行研究。
方法:检测斑马鱼从受精后4天到7dpf的行为轨迹,分析miR-196a-1和miR-196b对运动能力的影响。采用苏木精-伊红(HE)染色和透射电镜(TEM)检测斑马鱼背侧肌肉组织。通过显微计算机断层扫描(micro-CT)检测斑马鱼的骨组织。实时荧光定量PCR检测相关基因的表达水平,包括vcp,dpm1,acta1b,mylpfb,col1a1a,bmp8a,gdf6a,和fgfr3。
结果:行为测试表明,总的行为轨迹,运动时间,miR-196a-1和miR-196b基因敲除系斑马鱼幼虫的运动速度均下降。肌肉组织分析显示缺乏miR-196a-1和miR-196b的斑马鱼肌纤维结构异常,表现为肌纤维空泡变性,核内迁移,黑色素沉积,和炎性细胞浸润。骨CT检查显示骨密度和骨小梁数量减少。real-timePCR结果显示,vcp的表达水平,dpm1,gdf6a,miR-196b基因敲除组中fgfr3和col1a1a降低。dpm1,acta1b的表达水平,mylpfb,gdf6a,和col1a1a下降,与野生型组相比,miR-196b基因敲除组的fgfr3表达水平升高。
结论:miR-196a-1和miR-196b在肌纤维结构中起重要作用,骨矿物质密度,和骨小梁数量通过影响vcp的表达,dpm1,acta1b,mylpfb,gdf6a,fgfr3和col1a1a进而影响电机系统的功能。
The exertion of motor function depends on various tissues, such as bones and muscles. miR-196 has been widely studied in cancer and other fields, but its effect on bone and skeletal muscle is rarely reported. In order to explore the role of miR-196 family in bone and skeletal muscle, we used the previously successfully constructed miR-196a-1 and miR-196b gene knockout zebrafish animal models for research.
The behavioral trajectories of zebrafish from 4 days post-fertilization (dpf) to 7 dpf were detected to analyze the effect of miR-196a-1 and miR-196b on motor ability. Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were used to detect the dorsal muscle tissue of zebrafish. The bone tissue of zebrafish was detected by microcomputed tomography (micro-CT). Real-time PCR was used to detect the expression levels of related genes, including vcp, dpm1, acta1b, mylpfb, col1a1a, bmp8a, gdf6a, and fgfr3.
The behavioral test showed that the total behavioral trajectory, movement time, and movement speed of zebrafish larvae were decreased in the miR-196a-1 and miR-196b gene knockout lines. Muscle tissue analysis showed that the structure of muscle fibers in the zebrafish lacking miR-196a-1 and miR-196b was abnormal and was characterized by vacuolar degeneration of muscle fibers, intranuclear migration, melanin deposition, and inflammatory cell infiltration. Bone CT examination revealed decreased bone mineral density and trabecular bone number. The real-time PCR results showed that the expression levels of vcp, dpm1, gdf6a, fgfr3, and col1a1a were decreased in the miR-196b gene knockout group. The expression levels of dpm1, acta1b, mylpfb, gdf6a, and col1a1a were decreased, and the expression level of fgfr3 was increased in the miR-196b gene knockout group compared with the wild-type group.
miR-196a-1 and miR-196b play an important role in muscle fiber structure, bone mineral density, and bone trabecular quantity by affecting the expression of vcp, dpm1, acta1b, mylpfb, gdf6a, fgfr3, and col1a1a and then affect the function of the motor system.