Wu Xuan, Chen Chen, Han Tiaotiao, Bian Huiqin, Chen Jinnan, Hei Ruoxuan, Meng Ye, Wu Chenjing, Lu Yaojuan, Gu Junxia, Qiao Longwei, Zheng Qiping
Department of Laboratory Medicine, School of Medicine, Jiangsu University Zhenjiang 212013, Jiangsu, China.
Department of Clinical Diagnose, Tangdu Hospital, Air Force Medical University Xi'an 710000, Shaanxi, China.
Am J Transl Res. 2023 Jun 15;15(6):4006-4019. eCollection 2023.
Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene () expression via interaction with promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of cis-enhancer, in controlling gene expression and chondrocyte hypertrophic differentiation.
The potential regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation.
The potential binding factor of cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes and . Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1.
Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp cis-enhancer.
先前已证明多种转录因子（TFs）通过与启动子相互作用来控制肥大软骨细胞特异性小鼠X型胶原基因（）的表达。本研究旨在探讨顺式增强子潜在结合因子信号转导和转录激活因子5a（Stat5a）在控制基因表达和软骨细胞肥大分化中的作用及机制。
通过对150 bp顺式增强子进行转录因子亲和力预测（TRAP）分析来预测潜在的调节因子。通过qRT-PCR、蛋白质印迹和免疫组化分析筛选并验证Stat5a。将Stat5a siRNA或表达质粒转染到MCT和ATDC5细胞中，以敲低或过表达Stat5a，并研究Stat5a对软骨细胞肥大过程中基因表达的影响。进行双荧光素酶报告基因检测以探索Stat5a影响转录的机制。进行阿尔新蓝、碱性磷酸酶和茜素红染色，以及相关标记基因的qRT-PCR分析，以研究Stat5a对软骨细胞分化的作用及可能机制。
顺式增强子Stat5a和Col10a1的潜在结合因子在肥大软骨细胞和中均高表达且呈正相关。敲低Stat5a可降低Col10a1表达，而过表达Stat5a则增强肥大软骨细胞中Col10a1的表达，表明Stat5a是Col10a1的正向调节因子。从机制上讲，Stat5a可增强由启动子/增强子介导的报告基因活性。此外，Stat5a增加了ATDC5细胞碱性磷酸酶染色的强度以及包括Runx2在内的相关肥大标记基因的表达，这与Stat5a和Col10a1的表达一致。
我们的结果支持Stat5a可能通过与150 bp顺式增强子相互作用促进Col10a1表达和软骨细胞肥大分化。
