Wei Yihao, Qi Tiantian, Cao Siyang, Zhang Weifei, Yu Fei, Zeng Hui, Weng Jian
Department of Bone & Joint Surgery, Peking University Shenzhen Hospital, Shenzhen 518036, China.
National & Local Joint Engineering Research Center of Orthopaedic Biomaterials, Peking University Shenzhen Hospital, Shenzhen 518036, China.
Exp Biol Med (Maywood). 2023 Mar;248(6):469-480. doi: 10.1177/15353702231151963. Epub 2023 Feb 27.
In recent years, an increasing number of studies have reported that long non-coding RNAs (lncRNAs) play essential regulatory roles in myogenic differentiation. In this study, a specific LncRNA XLOC_015548 (Lnc000280) was identified. However, little research has explored its mechanism of action by constructing XLOC_015548 gene editing cell models. In this study, relevant sequences were obtained according to the RNA-seq results. Subsequently, XLOC_015548 knockdown and over-expression lentiviral vectors were constructed, and the C2C12 myoblast cell line was transfected to prepare the XLOC_015548 gene-edited myoblast model. The analysis revealed that over-expression of XLOC_015548 significantly promoted the proliferation and differentiation of myoblasts and the formation of myotubes, whereas the opposite result was obtained in the knockdown group. XLOC_015548 regulated myogenic differentiation and affected the expression of myogenic differentiation regulators such as Myod, myogenin, and MyHC. Regarding the signaling pathway, we found that XLOC_015548 correlated with the phosphorylation level of MAPK/MEK/ERK pathway proteins. And the degree of phosphorylation was positively correlated with the protein expression of myogenic differentiation regulators. In conclusion, a new gene-edited myoblast model was constructed based on the lncRNA regulator XLOC_015548. The cell experiments verified that XLOC_015548 had regulatory effects on muscle growth and myoblast differentiation. These findings provide a laboratory foundation for the clinical application of lncRNAs as regulatory factors in the treatment of disuse muscle atrophy.
近年来,越来越多的研究报道长链非编码RNA(lncRNAs)在肌源性分化中发挥重要的调节作用。在本研究中,鉴定出一种特异性的LncRNA XLOC_015548(Lnc000280)。然而,通过构建XLOC_015548基因编辑细胞模型来探究其作用机制的研究较少。在本研究中,根据RNA测序结果获得了相关序列。随后,构建了XLOC_015548敲低和过表达慢病毒载体,并转染C2C12成肌细胞系以制备XLOC_015548基因编辑的成肌细胞模型。分析显示,XLOC_015548的过表达显著促进了成肌细胞的增殖、分化以及肌管的形成,而敲低组则得到相反的结果。XLOC_015548调节肌源性分化,并影响Myod、肌细胞生成素和MyHC等肌源性分化调节因子的表达。关于信号通路,我们发现XLOC_015548与MAPK/MEK/ERK通路蛋白的磷酸化水平相关。并且磷酸化程度与肌源性分化调节因子的蛋白表达呈正相关。总之,基于lncRNA调节因子XLOC_015548构建了一种新的基因编辑成肌细胞模型。细胞实验证实XLOC_015548对肌肉生长和成肌细胞分化具有调节作用。这些发现为lncRNAs作为调节因子在废用性肌肉萎缩治疗中的临床应用提供了实验室基础。