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文俊儒, 谭震, 林玮民, 等. m6A结合蛋白YTHDC2对人骨髓间充质干细胞分化的影响[J]. 四川大学学报(医学版), 2021, 52(3): 402-408. DOI: 10.12182/20210560204
引用本文: 文俊儒, 谭震, 林玮民, 等. m6A结合蛋白YTHDC2对人骨髓间充质干细胞分化的影响[J]. 四川大学学报(医学版), 2021, 52(3): 402-408. DOI: 10.12182/20210560204
WEN Jun-ru, TAN Zhen, LIN Wei-min, et al. Role of m6A Reader YTHDC2 in Differentiation of Human Bone Marrow Mesenchymal Stem Cells[J]. Journal of Sichuan University (Medical Sciences), 2021, 52(3): 402-408. DOI: 10.12182/20210560204
Citation: WEN Jun-ru, TAN Zhen, LIN Wei-min, et al. Role of m6A Reader YTHDC2 in Differentiation of Human Bone Marrow Mesenchymal Stem Cells[J]. Journal of Sichuan University (Medical Sciences), 2021, 52(3): 402-408. DOI: 10.12182/20210560204

m6A结合蛋白YTHDC2对人骨髓间充质干细胞分化的影响

Role of m6A Reader YTHDC2 in Differentiation of Human Bone Marrow Mesenchymal Stem Cells

  • 摘要:
      目的  研究N6-腺苷酸甲基化(N6-methyladenosine, m6A)结合蛋白YTH结构域蛋白2(YTH domain-containing protein 2,YTHDC2)对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)成骨及成脂分化的调控。
      方法  通过小干扰RNA(siRNA)体外对hBMSCs进行YTHDC2基因表达的敲降,并进行成骨及成脂诱导分化,以研究YTHDC2敲降后hBMSCs分化表型的改变。利用碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红染色鉴定成骨活性和钙结节形成,尼罗红染色检测脂滴形成。利用荧光定量PCR(RT-qPCR)检测成骨和成脂相关基因的表达。通过RNA测序(RNA-seq)分析YTHDC2敲降后的转录组变化,探索YTHDC2调控hBMSCs分化的潜在机制。
      结果  敲降YTHDC2促进hBMSCs成骨分化中的ALP活性及钙结节形成,并显著上调成骨相关基因表达;同时降低了hBMSCs在成脂分化中的脂滴形成能力,并显著下调成脂相关基因表达。RNA-seq的基因富集分析显示YTHDC2与核糖体功能及mRNA翻译有关信号通路显著相关。
      结论  敲降YTHDC2可促进hBMSCs成骨分化,抑制成脂分化。敲降YTHDC2可能造成核糖体功能改变。

     

    Abstract:
      Objective  To study the regulatory effect of YTH domain-containing protein 2 (YTHDC2), a member of N6-methyladenosine (m6A) readers, on the osteogenic or adipogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
      Methods  YTHDC2 expression was knocked down by small interfering RNA (siRNA) in vitro. Osteogenic differentiation and adipogenic differentiation of hBMSCs were induced after YTHDC2 knockdown in order to study the changes in the differentiation phenotype of hBMSCs. Alkaline phosphatase staining (ALP staining) and alizarin red S staining were performed to examine osteogenic activity and calcium-nodular formation. Nile red staining was performed to examine lipid-droplet formation. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of osteogenesis and adipogenesis-related genes. RNA-sequencing was performed to identify the transcriptome changes after YTHDC2 knockdown and to explore the potential regulatory mechanism by which YTHDC2 regulated the diferentiation of hBMSCs.
      Results  In this study, we found that siRNA-induced YTHDC2 knockdown resulted in increased ALP activity and calcium-nodular formation of hBMSCs during osteogenic differentiation, and significantly upregulated the expression of osteogenesis-related genes. In addition, the lipid-droplet formation capacity of hBMSCs was decreased during adipogenic differentiation. The expression of adipogenesis-related genes was significantly down-regulated. Gene-set enrichmen analysis of RNA-seq data showed that YTHDC2 was significantly correlated with ribosome function and mRNA-translation-related signaling pathways.
      Conclusion  The findings indicate that YTHDC2 knockdown can promote the osteogenic differentiation of hBMSCs and inhibit the adipogenic differentiation. YTHDC2 knockdown may cause changes in ribosome function.

     

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