Zhu Jun, Fu Qiwei, Shao Jiahua, Qian Qirong, Zhou Yiqin
Department of Joint Surgery and Orthopedic Medicine, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, People's Republic of China.
J Tissue Eng Regen Med. 2021 May;15(5):487-502. doi: 10.1002/term.3189. Epub 2021 Apr 7.
Circ_ATRNL1 is significantly highly expressed in cartilage tissues of patients with osteoarthritis. This study explored the role and mechanism of circ_ATRNL1 in cartilage differentiation of human adipose-derived mesenchymal stem cells (hAMSCs). hAMSCs were isolated and identified by flow cytometry. The degree of chondrocyte and adipogenic differentiation was assessed using Alcian blue staining and Oil Red O staining, respectively. The expressions of cartilage- and adipogenic-related genes, circ_ATRNL1, and SOX9 were detected by reverse transcription quantitative polymerase chain reaction. The correlation between SOX9 and circ_ATRNL1 was analyzed using Pearson test. Bioinformatics and luciferase analysis were used to detect the overlapped target miRNAs of circ_ATRNL1 and SOX9. The role of circ_ATRNL1/miRNA/SOX9 was examined using functional rescue assays. hAMSCs were identified as CD90-, CD105-, and CD44-positive. The degree of cartilage differentiation of hAMSCs was significantly enhanced after 2 weeks. Cartilage-related genes, circ_ATRNL1 and SOX9, were significantly upregulated, and positively correlated with each other. Circ_ATRNL1 overexpression enhanced hAMSC proliferation and differentiation into chondrogenesis, and promoted the expressions of COL2, Aggrecan, and SOX9. Overexpression of circ_ATRNL1 inhibited the adipogenic differentiation of hAMSCs and the expressions of adipogenic-related genes. miR-145-5p was a target miRNA for circ_ATRNL1 and SOX9. miR-145-5p mimic inhibited hAMSC differentiation toward cartilage, and inhibited the expression of cartilage-related factors. miR-145-5p mimic effectively reversed the regulating effect of circ_ATRNL1 on hAMSCs. Circ_ATRNL1 regulates the promotion of SOX9 expression to promote chondrogenic differentiation of hAMSCs mediated by miR-145-5p.
环状ATRNL1在骨关节炎患者的软骨组织中显著高表达。本研究探讨了环状ATRNL1在人脂肪间充质干细胞（hAMSCs）软骨分化中的作用及机制。通过流式细胞术分离并鉴定hAMSCs。分别使用阿尔辛蓝染色和油红O染色评估软骨细胞和成脂分化程度。通过逆转录定量聚合酶链反应检测软骨和成脂相关基因、环状ATRNL1和SOX9的表达。使用Pearson检验分析SOX9与环状ATRNL1之间的相关性。采用生物信息学和荧光素酶分析检测环状ATRNL1和SOX9的重叠靶标miRNA。使用功能挽救试验检测环状ATRNL1/miRNA/SOX9的作用。hAMSCs被鉴定为CD90、CD105和CD44阳性。2周后hAMSCs的软骨分化程度显著增强。软骨相关基因、环状ATRNL1和SOX9显著上调，且彼此呈正相关。环状ATRNL1过表达增强了hAMSCs的增殖和向软骨形成的分化，并促进了COL2、聚集蛋白聚糖和SOX9的表达。环状ATRNL1过表达抑制了hAMSCs的成脂分化和成脂相关基因的表达。miR-145-5p是环状ATRNL1和SOX9的靶标miRNA。miR-145-5p模拟物抑制hAMSCs向软骨的分化，并抑制软骨相关因子的表达。miR-145-5p模拟物有效逆转了环状ATRNL1对hAMSCs的调节作用。环状ATRNL1通过调节SOX9表达的促进作用，介导miR-145-5p促进hAMSCs的软骨分化。
