A Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of the Ministry of Education and Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China.
The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China.
Cells. 2020 Feb 15;9(2):449. doi: 10.3390/cells9020449.
Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 5'- and 3'-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 (), namely, v1 and v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript v2. Functional analyses showed that overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor (), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of . Dual luciferase reporter assay showed that could attenuate the decrease of luciferase activity of induced by miR-423-5p in a dose-dependent manner. overexpression could relieve the inhibitory effect on and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among , miR-423-5p, and in porcine satellite cell differentiation. This novel role of may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis.
虽然已经在猪的生长和发育过程中鉴定出了数千种长链非编码 RNA(lncRNA),但功能 lncRNA 的调控机制尚未得到很好的探索。在这项研究中,我们使用 5'和 3'快速扩增 cDNA 末端(RACE)实验,获得了两种不同的长链非编码 RNA 母系表达基因 3()变体,即 v1 和 v2,它们在猪骨骼肌和猪卫星细胞分化的早期均高度表达。此外,我们鉴定了核心转录本 v2。功能分析表明,过表达可以有效地将成肌细胞阻滞在 G1 期,抑制 DNA 复制,并促进成肌细胞分化,而敲低则产生相反的效果。有趣的是,血清反应因子()的表达显著增加和减少,在 mRNA 和蛋白水平上,与各自的过表达和敲低。双荧光素酶报告基因实验表明,在剂量依赖性方式下,能够减弱 miR-423-5p 诱导的荧光素酶活性的降低。过表达可以缓解 miR-423-5p 对和成肌细胞分化的抑制作用。此外,RNA 免疫沉淀分析的结果表明,在猪卫星细胞分化过程中,可能作为 miR-423-5p 的 ceRNA 发挥作用。我们的研究结果初步建立了猪卫星细胞分化过程中、miR-423-5p 和之间的新联系。在猪肌发生中,lncRNA 的这种新作用可能为理解 lncRNA 的分子调控提供新的思路。