Decorin knockdown affects the gene expression profile of adhesion, growth and extracellular matrix metabolism in C-28/I2 chondrocytes.

Mengying Wang,Hui Wang,Yinan Liu,Meng Zhang,Jinghong Chen,Ying Zhang,Zhengzheng Li,Yiping Feng,Dragana Nikitovic

doi:10.1371/journal.pone.0232321

Abstract

Decorin is a member of small leucine-rich proteoglycan family, which is involved in multiple biological functions mainly as a structural and signaling molecule, and disturbances in its own metabolism plays a crucial role in the pathogenesis of osteoarthropathy. In this study, we aim to further explore the biological function of decorin and their role in human chondrocyte cell line, C28/I2. Lentivirus-mediated shRNA was applied to down-regulate decorin expression in C28/I2 chondrocytes. Effect of decorin knockdown on gene expression profiles was determined by RNA sequencing followed by bioinformatics analysis. MTT, adhesion assays and flow cytometry were used to investigate the effect of decorin knockdown on cell proliferation, adhesion, and apoptosis. sGAG content in the culture medium was determined by DMMB assay. Stably transfected C28/I2 cells were seeded onto the cancellous bone matrix gelatin (BMG) to construct tissue-engineered cartilage. The histological patterns were evaluated by H&E and Toluidine blue staining. In this study, 1780 differentially expressed genes (DEGs) including 864 up-regulated and 916 down-regulated genes were identified using RNA-Seq. The reliability of the gene expression was further verified by qRT-PCR. GO and KEGG pathway enrichment analysis revealed diverse cellular processes were affected by decorin silencing such as: cell adhesion, growth, and metabolism of extracellular matrix. In addition, we confirmed that down-regulation of decorin significantly suppressed cell proliferation and adhesion and induced apoptosis. The sGAG content in the media was significantly increased after decorin silencing. Engineered articular tissues in the decorin knockdown group exhibited cartilage destruction and proteoglycan loss as evidenced by H&E and Toluidine blue stains. Overall, this combined data helps to provide a comprehensive understanding of the roles of decorin following its knockdown in C28/I2 cells.

Highlights

Decorin is a prominent member of the class I small leucine-rich proteoglycan (SLRP) family in the extracellular matrix (ECM), and consists of a core protein and a single chondroitin/dermatan sulfate glycosaminoglycan (GAG) side chain at the N-terminal
Decorin was initially defined as structural components modulating the synthesis, organization, and assembly of collagen fibrils [1,2], but with time has evolved as a versatile proteoglycan, involved in various biological processes such as cell proliferation, differentiation [3], matrix adhesion [4], autophagy [5], inflammation and immunity [6], angiogenesis [7], tumorigenesis [8], osteoarthritis and osteoporosis [9,10,11], and fibrosis [12]
By using integrated bioinformatics analysis, we further identified the differentially expressed genes (DEGs) and pathways regulated by decorin, which could improve our understanding of the underlying molecular events regulated by these genes in osteoarticular diseases

Summary

Introduction

Decorin is a prominent member of the class I small leucine-rich proteoglycan (SLRP) family in the extracellular matrix (ECM), and consists of a core protein and a single chondroitin/dermatan sulfate glycosaminoglycan (GAG) side chain at the N-terminal. Decorin is highly expressed in cartilage, is a key matrix constituent for the extracellular matrix and is involved in proper cartilage biomechanical functions. In osteoarthritis (OA), the small proteoglycans (PG), decorin and biglycan, are lost from the surface of articular cartilage; their contents increase in the deeper parts of the cartilage [17]. Extensive degradation of both large and small PGs, such as decorin and biglycan were found in cartilages of patients suffering from OA and rheumatoid arthritis (RA) [9,11,18]. Recent studies have reported that decorin deficiency leads to the altered ECM biomechanical properties and cartilage stiffness, and decorin-null mice are less prone to develop OA after forced exercise [20]

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