Transcriptome Analysis of Platelet-Rich Plasma-Treated Osteoarthritic Chondrocyte

Abstract

As a blood-derived biomaterial, platelet-rich plasma (PRP) has been considered a potential therapy and tried in knee and hip osteoarthritis with beneficial effects as an anti-inflammatory and potent regenerative agent. To better understand the substantial effect of PRP on chondrocytes in an inflammatory environment, we analyzed the transcriptome profile by RNA sequencing (RNA-seq) after PRP administration in IL-1β-treated osteoarthritic chondrocytes which were isolated from human knee articular cartilage tissue. A total of 24,424 genes were analyzed, and significant changes in expression were observed for 226 genes in the control (CTL) versus IL-1β group and 300 genes in the IL-1β versus IL-1β+PRP group. The Top 20 significantly upregulated and downregulated genes and the major altered genes in nine categories that are closely related to chondrocyte physiology were analyzed, and the expression of several important genes in each category was evaluated by qRT-PCR and western blot analysis. Our study revealed that the PRP, at the gene expression level, has apparent anti-inflammatory, cell proliferative, and regenerative activities in chondrocytes in the presence of IL-1β, which mimic an osteoarthritic environment. Identifying potent molecules that regulate cartilage physiology represents a promising therapeutic approach for suppressing cartilage degeneration, especially that caused by inflammation-induced osteoarthritis.

Figure 1

Isolation of human chondrocytes and their differentiation and proliferation by PRP. (a) Overview of chondrocytes isolation procedure. (b) Evaluation of isolated chondrocyte by staining with glycosaminoglycan, anti-collagen type II antibody, and anti-MMP3 antibody in the presence of IL-1β. (c) Overview of PRP isolation procedure. (d) Microscopic morphology of chondrocytes after PRP administration in the presence of IL-1β.

Figure 2

Transcriptome analysis by RNA sequencing (RNA-seq). (a) Overview of RNA-seq process (n = 6/group). (b) Left panel: Venn diagram depicting the number of genes up, down, and contraregulated among the significant genes for each group; PRP/control (CTL), IL-1β/CTL, and IL-1β+PRP/IL-1β. Right panel: scatter plot showing the distribution of log2 (normalized data) for each gene between two groups (red: upregulation, green: downregulation) (n = 6/group). (c) Percentages of total significant genes according to 18 categories for each group; PRP/control (CTL), IL-1β/CTL, and IL-1β+PRP/IL-1β (n = 6/group).

Figure 3

Differential expression patterns in hierarchical clustering and the STRING network for the inflammatory response category. (a) Left panel: different color index showing gene expression fold between two groups. Right panel: information for interpreting the STRING network. (b) Upper panels: differential expression patterns in hierarchical clustering of the altered genes in the inflammatory response category for IL-1β/CTL and IL-1β+PRP/IL-1β group. Lower panel: the STRING network for each altered gene (n = 6/group).

Figure 4

Differential expression patterns in hierarchical clustering and the STRING network for the cartilage development and cell proliferation categories. (a) Differential expression patterns in hierarchical clustering of the altered genes in the cartilage development category for IL-1β/CTL and IL-1β+PRP/IL-1β group. The lower panel shows the STRING network for each altered gene (n = 6/group). (b) Differential expression patterns in hierarchical clustering of the altered genes in the cell proliferation category for IL-1β/CTL and IL-1β+PRP/IL-1β group. Lower panel: the STRING network for each altered gene (n = 6/group).

Figure 5

Differential expression patterns in hierarchical clustering and the STRING network for the cell differentiation category. (a) Differential expression patterns in hierarchical clustering of the altered genes in the cell differentiation category for IL-1β/CTL and IL-1β+PRP/IL-1β group. (b) The STRING network for each altered gene (n = 6/group).

Figure 6

Validation of gene expression using real-time qPCR and western blot analyses. mRNA expression levels of the representative altered genes in the (a) inflammatory response, (b) chondrocyte homeostasis, (c) cell proliferation, and (d) cell differentiation categories for the validation of gene expression using quantitative real-time PCR analysis. The p values between two compared groups (IL-1β/CTL and IL-1β+PRP/IL-1β) in (a)–(d) were all < 0.05. (e) Western blot analysis for the representatively selected genes from the results of qRT-PCR analysis. Densitometric analyses of the western blots are shown on the right. Data represent means ± standard errors of the means. ⁣^∗p < 0.05.