Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of the Ministry of Education and Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, 430070, Wuhan, China.
Cell Death Dis. 2020 Feb 24;11(2):148. doi: 10.1038/s41419-020-2341-3.
miRNAs reportedly participate in various biological processes, such as skeletal muscle proliferation and differentiation. However, the regulation of differentially expressed (DE) miRNAs and their function in myogenesis remain unclear. Herein, miRNA expression profiles and regulation during C2C12 differentiation were analyzed in relation to chromatin states by RNA-seq, ATAC-seq, and ChIP-seq. We identified 19 known and nine novel differentially expressed miRNAs at days 0, 1, 2, and 4. The expression of the differentially expressed miRNAs was related to the chromatin states of the 113 surrounding open chromatin regions defined by ATAC-seq peaks. Of these open chromatin regions, 44.25% were colocalized with MyoD/MyoG binding sites. The remainder of the above open chromatin regions were enriched with motifs of the myoblast-expressed AP-1 family, Ctcf, and Bach2 transcription factors (TFs). Additionally, the target genes of the above differentially expressed miRNAs were enriched primarily in muscle growth and development pathways, especially the Hippo signaling pathway. Moreover, via combining a loss-of-function assay with Q-PCR, western blotting, and immunofluorescence, we confirmed that the Hippo signaling pathway was responsible for C2C12 myoblast differentiation. Thus, our results showed that these differentially expressed miRNAs were regulated by chromatin states and affected muscle differentiation through the Hippo signaling pathway. Our findings provide new insights into the function of these differentially expressed miRNAs and the regulation of their expression during myoblast differentiation.
miRNAs 据报道参与各种生物过程，如骨骼肌增殖和分化。然而，差异表达 (DE) miRNAs 的调节及其在成肌中的功能仍不清楚。在此，通过 RNA-seq、ATAC-seq 和 ChIP-seq 分析了 C2C12 分化过程中 miRNA 表达谱和与染色质状态的关系。我们在第 0、1、2 和 4 天鉴定了 19 个已知和 9 个新的差异表达 miRNA。差异表达 miRNA 的表达与 ATAC-seq 峰定义的 113 个周围开放染色质区域的染色质状态有关。在这些开放染色质区域中，44.25%与 MyoD/MyoG 结合位点共定位。上述开放染色质区域的其余部分富含成肌细胞表达的 AP-1 家族、Ctcf 和 Bach2 转录因子 (TF) 的基序。此外，上述差异表达 miRNA 的靶基因主要富集在肌肉生长和发育途径中，特别是 Hippo 信号通路。此外，通过结合功能丧失测定、Q-PCR、western blot 和免疫荧光，我们证实 Hippo 信号通路负责 C2C12 成肌细胞分化。因此，我们的研究结果表明，这些差异表达的 miRNA 受染色质状态调节，并通过 Hippo 信号通路影响肌肉分化。我们的研究结果为这些差异表达的 miRNA 的功能及其在成肌细胞分化过程中的表达调节提供了新的见解。
