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A critical role for thrombin in regulating chondrocytes catabolism through RNA-Seq and experimental validation

ABSTRACT: A critical role for thrombin in regulating chondrocytes catabolism through RNA-Seq and experimental validation

PROVIDER: PRJNA762871 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15871678_1.fastq.gz	Fastqsanger.gz
	SRR15871678_2.fastq.gz	Fastqsanger.gz
	SRR15871679_1.fastq.gz	Fastqsanger.gz
	SRR15871679_2.fastq.gz	Fastqsanger.gz
	SRR15871680_1.fastq.gz	Fastqsanger.gz

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Project description: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.

Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-R´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Keywords: Leukotriene Transcriptome, Thrombin Transcriptome, HUVEC, Immediate-Early Gene Expression, Cysteinyl Leukotriene 2 Receptor Gene Signature in HUVEC

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A critical role for thrombin in regulating chondrocytes catabolism through RNA-Seq and experimental validation

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