Liu Feng, Song De-Ye, Huang Jun, Yang Hong-Qi, You Di, Ni Jiang-Dong
Department of Orthopedics, The Second Xiangya Hospital of Central South University, No.139 Renminzhong Road, Changsha, 410011, Hunan, People's Republic of China.
Department of Orthopedics, The Third Hospital of Changsha, Changsha, 410015, Hunan, People's Republic of China.
Mol Med. 2021 Feb 5;27(1):12. doi: 10.1186/s10020-021-00272-9.
Osteoarthritis (OA) is the most common articular disorder, leading to joint malfunction and disability. Although the incidence of OA is increasing globally, the treatment of OA is very limited. LncRNA CIR has been implicated in OA through unclear mechanisms. Here, we investigated the role of lncRNA CIR in chondrogenic differentiation.
Human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) were obtained from human umbilical cords. Flow cytometry was used to analyze the surface markers of hUC-MSCs. Various culture conditions and corresponding staining assays were employed to assess the differentiation abilities of hUC-MSC. qRT-PCR, western blot, and immunostaining were used to measure expression levels of related genes and proteins such as lncRNA CIR, ATOH8, EZH2, and H3K27me3. RNA immunoprecipitation assay, biotin pull-down, and chromatin immunoprecipitaion assay were performed to analyze the interactions of lncRNA CIR, EZH2, H3K27me3 and ATOH8 promoter.
hUC-MSCs exhibited MSCs features and could differentiate into chondrocytes under specific conditions. LncRNA CIR was downregulated while ATOH8 was upregulated during the chondrogenic differentiation of hUC-MSCs. Knockdown lncRNA CIR or overexpression of ATOH8 promoted chondrogenic differentiation. Further, lncRNA CIR bound to EZH2 and repressed ATOH8 expression via EZH2-mediated H3K27me3, which promotes the methylation of ATOH8. Inhibition of ATOH8 reversed the effects of knockdown lncRNA CIR on chondrogenic differentiation.
LncRNA CIR suppresses chondrogenic differentiation of hUC-MSCs. Mechanistically, lncRNA CIR could inhibit ATOH8 expression that functions to promote chondrogenic differentiation through EZH2-mediated epigenetic modifications.
骨关节炎（OA）是最常见的关节疾病，可导致关节功能障碍和残疾。尽管OA的发病率在全球范围内呈上升趋势，但OA的治疗方法非常有限。长链非编码RNA（lncRNA）CIR通过不明机制与OA相关。在此，我们研究了lncRNA CIR在软骨形成分化中的作用。
从人脐带中获取人脐带间充质干细胞（hUC-MSCs）。采用流式细胞术分析hUC-MSCs的表面标志物。采用各种培养条件和相应的染色试验评估hUC-MSC的分化能力。采用qRT-PCR、蛋白质印迹法和免疫染色法检测lncRNA CIR、ATOH8、EZH2和H3K27me3等相关基因和蛋白质的表达水平。进行RNA免疫沉淀试验、生物素下拉试验和染色质免疫沉淀试验，以分析lncRNA CIR、EZH2、H3K27me3与ATOH8启动子之间的相互作用。
hUC-MSCs表现出间充质干细胞特征，并在特定条件下可分化为软骨细胞。在hUC-MSCs的软骨形成分化过程中，lncRNA CIR表达下调，而ATOH8表达上调。敲低lncRNA CIR或过表达ATOH8可促进软骨形成分化。此外，lncRNA CIR与EZH2结合，并通过EZH2介导的H3K27me3抑制ATOH8表达，从而促进ATOH8的甲基化。抑制ATOH8可逆转敲低lncRNA CIR对软骨形成分化的影响。
lncRNA CIR抑制hUC-MSCs的软骨形成分化。机制上，lncRNA CIR可通过EZH2介导的表观遗传修饰抑制ATOH8的表达，而ATOH8的功能是促进软骨形成分化。
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