Tang Xiang, Su Xianghui, Zhong Zhuohui, Wen Canliang, Zhang Tiansong, Zhu Yali
Department of Gynecology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University Guangzhou 510623, China.
Am J Transl Res. 2019 Aug 15;11(8):5150-5161. eCollection 2019.
We investigated the molecular mechanisms involved in transforming growth factor beta 1 (TGF-β1)-induced myogenic stem cell differentiation to smooth muscle cells. We isolated muscle-derived stem cells (MDSCs) from gastrocnemius muscles following their identification by immunohistochemistry analysis of desmin and flow cytometry analysis of SCA-1, CD34, and CD45. MDSCs at passage 3 (PP3) were cultured to examine the effects of MDSC induction. Gene ontology and KEGG pathway analyses were performed to analyze these differentially expressed genes. Reduced representation bisulfite sequencing was performed in TGF-β1-treated and untreated cells to evaluate differences in the methylation status and analyze the chromosomal distribution of differentially methylated sites (DMSs). Significant morphological changes to cells were observed at PP3, and most PP3 cells were positive for desmin and SCA-1, and were confirmed to be MDSCs. Results of western blot and immunohistochemistry analyses suggested that expressions of a-SMA and CNN1 significantly increased after treatment with TGF-β1. Global transcriptome analysis identified 1996 differentially expressed genes (MSC_TGFβ1/MSC_NC). Results of methylome analysis indicated that there were more hypermethylation sites in the untreated group than in the TGF-β1-treated group. Most DMSs were hypermethylated, whereas a small portion was hypomethylated. The chromosomal distribution of DMSs indicated that chromosome 1 had the highest proportion of DMSs, whereas the Y chromosome had the fewest DMSs. Sud2, Pcdh19, and Nat14 are potential core genes involved in cell differentiation. These results may explain the mechanisms of cell differentiation and provide useful information regarding diseases such as pelvic organ prolapse.
我们研究了转化生长因子β1（TGF-β1）诱导肌源性干细胞分化为平滑肌细胞所涉及的分子机制。我们从腓肠肌中分离出肌肉衍生干细胞（MDSCs），之后通过结蛋白的免疫组织化学分析以及SCA-1、CD34和CD45的流式细胞术分析对其进行鉴定。培养第3代（PP3）的MDSCs以检测MDSC诱导的效果。进行基因本体论和KEGG通路分析以分析这些差异表达基因。在TGF-β1处理和未处理的细胞中进行简化代表性亚硫酸氢盐测序，以评估甲基化状态的差异并分析差异甲基化位点（DMSs）的染色体分布。在PP3观察到细胞有明显的形态变化，并且大多数PP3细胞结蛋白和SCA-1呈阳性，证实为MDSCs。蛋白质免疫印迹和免疫组织化学分析结果表明，TGF-β1处理后α-SMA和CNN1的表达显著增加。全转录组分析鉴定出1996个差异表达基因（MSC_TGFβ1/MSC_NC）。甲基化组分析结果表明，未处理组的高甲基化位点比TGF-β1处理组更多。大多数DMSs是高甲基化的，而一小部分是低甲基化的。DMSs的染色体分布表明，1号染色体的DMSs比例最高，而Y染色体的DMSs最少。Sud2、Pcdh19和Nat14是参与细胞分化的潜在核心基因。这些结果可能解释细胞分化的机制，并为诸如盆腔器官脱垂等疾病提供有用信息。
