GSE184026
A critical role for thrombin in regulating chondrocytes catabolism through RNA-Seq and experimental validation
Purpose: To reveal the effects of thrombin on the proliferation, morphology, and gene expression in chondrocytes. Methods: Rat primary chondrocytes were cultured with RS, RP and RPT for 24hours, and RNA was extracted for sequencing. The chondrocytes' morphology was investigated under inverted microscope. Chondrocyte's proliferation was evaluated by Edu staining, CCK8 methods and PPI network of proliferation-associated genes. The differential gene expression was visualized using a volcano plot. The variance in expression levels of the genes was visualized by the heatmap. GO-based functional annotation clustering analysis was performed on the upregulated and downregulated DEGs, which included biological process, cellular component, and molecular function. The endoplasmic reticulum of chondrocytes from three groups were labeled by molecular probes. The expression levels of Col2a1 and proteoglycan synthesis were detected by cellular immunofluorescence and Safranin O staining separately. The expression levels of Lox were determined by immunocytochemistry staining. The following genes including Comp, Eln, P3H1 and Colgalt1 were examined by RT-qPCR. The expression of MMPs and Sox9 were evaluated by Western Blot. Further analysis of PPI network was performed, and the corresponding modules were selected. Results: As compared with RP group, chondrocytes proliferation was obvious and normal chondrocytes quickly transformed into fibroblast-like chondrocytes in the presence of RS and RPT. we found that 727 genes were up-regulated, and 1162 genes were down-regulated in Venn diagram through analyzing differentially expressed genes (DEGs). The results of GO analyses showed that both the expression of ECM and MMPs were significantly influenced by RS and RPT. Both the labeled endoplasmic reticulum and the expression of representative genes analyzed from the results of RNA sequencing confirmed that chondrocytes were dominated by catabolism. The expression of ECM, MMPs, inflammatory factors and chemokines were verified at the protein level or at the RNA level which agreed with PPI network and functional modules. Conclusions: Thrombin stimulated the proliferation and promoted the morphology transformation towards fibrotic chondrocytes. Thrombin improved the chondrocytes' catabolism by regulating the expression of the key enzymes which were responsible for ECM synthesis. Meanwhile thrombin increased the expression of MMPs and chemokines that aimed at extracellular ECM degradation. Overall design: Rat primary chondrocytes were cultured with Rat Serum (RS  group), Rat Plasma  (RP  group), and Rat plasma plus thrombin (RPT  group) for 24hours, and RNA was extracted for sequencing. Further experiments were performed to verify the results of RNA sequencing.