OCT4 通过 DNA 甲基转移酶下调 p21 来维持人毛囊间充质干细胞的自我更新并逆转衰老。
OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases.
机构信息
The Key Laboratory of Pathobiology, Ministry of Education, Department of Pathology, College of Basic Medical Sciences, Jilin University, 126 Xinmin Avenue, Changchun, 130021, China.
Cell Processing Section, Department of Transfusion, Clinical Center, National Institutes of Health, Bethesda, MD, 20892, USA.
出版信息
Stem Cell Res Ther. 2019 Jan 15;10(1):28. doi: 10.1186/s13287-018-1120-x.
BACKGROUND
Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved.
METHODS
A lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCs to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected.
RESULTS
The overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCs and hHFMSCs, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCs and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCs, and triggered cell senescence.
CONCLUSIONS
OCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.
背景
自我更新依赖于以 OCT4 为中心的内在基因调控网络，以及由 OCT4 调控的非典型细胞周期 G1/S 转变。p21 是一个与自我更新和衰老标志物负相关的基因，是普遍细胞周期蛋白依赖性激酶抑制剂（CDKIs）的成员，在 G1/S 转变的调控中起着关键作用。p21 的表达可以被 OCT4 靶向的 DNA 甲基转移酶（DNMTs）调控，DNMTs 在基因调控和维持多能性方面发挥着不同的作用。本研究旨在确定 OCT4 在人毛囊间充质干细胞（hHFMSCs）自我更新和衰老中的调控作用，并探讨其涉及的分子机制。
方法
使用慢病毒载体过表达 OCT4。研究 OCT4 对 hHFMSCs 自我更新和衰老的影响。接下来进行下一代测序（NGS），以鉴定在此过程中 OCT4 的下游基因。甲基化特异性 PCR（MSP）分析用于测量 p21 启动子区域的甲基化水平。在 hHFMSCs 中过表达 p21，以检验其对 OCT4 的下游影响。通过 ChIP assay 检测 OCT4 对 DNMTs 的调控作用。使用 5-aza-dC/zebularine 抑制 DNMTs 的表达，然后检测 hHFMSCs 的自我更新特性和衰老。
结果
OCT4 的过表达促进了 hHFMSCs 的增殖、细胞周期进程和成骨分化能力。由于 OCT4 的异位表达，hHFMSCs 的细胞衰老明显受到抑制。通过 NGS，我们在 hHFMSCs 和 hHFMSCs 之间鉴定出 2466 个差异表达基因（DEGs），包括下调的 p21。p21 的过表达消除了 hHFMSCs 的增殖和成骨分化能力，并促进了细胞衰老。OCT4 增强了 DNMT 基因的转录，导致 p21 启动子的甲基化水平升高。DNMTs 的抑制作用逆转了 OCT4 诱导的 p21 减少，耗尽了 hHFMSCs 的自我更新能力，并引发了细胞衰老。
结论
OCT4 通过上调 DNMTs 抑制 p21 的表达，维持 hHFMSCs 的自我更新能力，并逆转衰老。
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