Identification of thrombin as a key regulator of chondrocyte catabolic activity through RNA-Seq and experimental verification
Identification of thrombin as a key regulator of chondrocyte catabolic activity through RNA-Seq and experimental verification
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通过 RNA-Seq 和实验验证鉴定凝血酶是软骨细胞分解代谢活性的关键调节剂
DOI：
10.1016/j.gene.2022.146327
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发表时间：
2022
期刊：
影响因子：
3.5
通讯作者：
Dao-Fang Ding
中科院分区：
文献类型：
--
作者：
Xi-Chen Wu;Zhi-Heng Zhu;Jun-Peng Zhang;Fu-Ming Shao;Jing-Qiu Peng;Yan Chen;Xue-Zong Wang;Wen-Yao Li;Yue-Long Cao;Wei Feng;Jian-Guang Xu;Dao-Fang Ding
本研究旨在探讨凝血酶与软骨细胞分解代谢活性的关系。用大鼠血清(RS)、大鼠血浆(RP)或大鼠血浆加凝血酶(RPT)培养原代大鼠软骨细胞24小时。然后进行RNA测序。通过EDU摄取、CCK-8检测和增殖相关基因的蛋白质-蛋白质相互作用(PPI)网络分析细胞增殖情况。热图被用来可视化基因表达的差异。对上调和下调的差异表达基因进行基因本体论(GO)浓缩分析。用分子探针标记三组软骨细胞的内质网。免疫荧光和藏红花O染色检测II型胶原(Col2a1)表达和蛋白多糖合成，免疫细胞化学检测Lox表达。用定量聚合酶链式反应(QPCR)检测细胞外基质(ECM)成分和趋化因子合成成熟相关酶的表达，用Western blotting检测基质金属蛋白酶(MMPs)水平。通过进一步的PPI网络分析，选择了相关的结节。根据维恩图在不同组之间的比较，共有727个和1162个基因上调和下调。因此，凝血酶能够促进软骨细胞的增殖和向纤维化形态的转变，同时上调与ECM降解有关的MMPs和趋化因子。此外，凝血酶还降低了参与ECM合成和成熟的酶的表达。
The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein–protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.