Peng Shujun, Song Chengchuang, Li Hui, Cao Xiukai, Ma Yilei, Wang Xiaogang, Huang Yongzhen, Lan Xianyong, Lei Chuzhao, Chaogetu Buren, Chen Hong
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Animal Disease Control Center of Haixi Mongolian and Tibetan Autonomous Prefecture, Delingha 817000, China.
Mol Ther Nucleic Acids. 2019 Jun 7;16:481-493. doi: 10.1016/j.omtn.2019.03.009. Epub 2019 Apr 9.
Myogenesis is a complex and precisely orchestrated process that is highly regulated by several non-coding RNAs and signal pathways. Circular RNAs (circRNAs) represent a novel subclass of endogenous non-coding RNAs that have been identified in multiple species and tissues and play a vital role in post-transcriptional regulation in eukaryotes, but the precise molecular mechanism of action remains largely unknown. Here, we screened a candidate circRNA derived from the SNX29 gene, termed circSNX29 from our previous circRNAs sequencing data of bovine skeletal muscle, and further characterized its regulation and function during muscle development. The overexpression of circSNX29 facilitated myoblasts differentiation and inhibited cell proliferation. Computational analysis using RNAhybrid showed the potential for circSNX29 to sponge to miR-744 with nine potential binding sites. We tested this via a luciferase screening assay and found that circSNX29 directly interacted with miR-744 and downregulation of miR-744 efficiently reversed the suppression of Wnt5a and CaMKIIδ. Importantly, through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, Fluo-4, AM, cell permeant-calcium ion fluorescent probing, and western blotting assays, we found that overexpression of Wnt5a and circSNX29 activated the non-canonical Wnt5a/Ca pathway. Overall, the evidence generated by our study elucidates the regulatory mechanisms of circSNX29 to function as a sponge for miRNA-744 in bovine primary myoblasts.
肌生成是一个复杂且精确编排的过程，受到多种非编码RNA和信号通路的高度调控。环状RNA（circRNA）是内源性非编码RNA的一个新亚类，已在多种物种和组织中被鉴定出来，并在真核生物的转录后调控中发挥重要作用，但其确切的分子作用机制仍 largely未知。在这里，我们从牛骨骼肌先前的circRNA测序数据中筛选出一个源自SNX29基因的候选circRNA，命名为circSNX29，并进一步表征了其在肌肉发育过程中的调控和功能。circSNX29的过表达促进了成肌细胞的分化并抑制了细胞增殖。使用RNAhybrid进行的计算分析表明circSNX29有潜力作为海绵吸附miR - 744，具有九个潜在结合位点。我们通过荧光素酶筛选试验对此进行了测试，发现circSNX29直接与miR - 744相互作用，miR - 744的下调有效地逆转了Wnt5a和CaMKIIδ的抑制作用。重要的是，通过京都基因与基因组百科全书（KEGG）通路富集分析、Fluo - 4、AM、细胞渗透性钙离子荧光探针和蛋白质印迹分析，我们发现Wnt5a和circSNX29的过表达激活了非经典Wnt5a/Ca通路。总体而言，我们的研究产生的证据阐明了circSNX29在牛原代成肌细胞中作为miRNA - 744海绵发挥功能的调控机制。
