3.4 PPI network construction
According to the information in STRING and Cytoscape databases, the PPI relationships of top 30 upregulated and downregulated DEGs were obtained (Fig. 4). The hub genes included matrix metalloproteinase (MMP)1, MMP3, MMP13, PTGS2, and so on. Among these genes, MMP1, MMP3, and MMP13 possessed the highest node degree.
3.5 Venn diagram containing the top 30 DEGs and genes from existed related studies
Many related studies have reported some genes involved in OA or developmental chondrogenesis. Cross-validation containing the top 30 DEGs and genes from related studies would be helpful for further research of OA. Among the top 30 upregulated and downregulated DEGs, there were 15 genes have been reported to be related to OA or developmental chondrogenesis (Fig. 5).
OA is a common chronic disease that afflicts the middle-aged and elderly individuals. According to statistics, the probability of this disease in male is approximately 40% while in female it is around 47%. Moreover, the incidence of people over 50 years old is rapidly increasing over time. Osteoarthritis is characterized by a decrease in articular cartilage tissue, thickening of subchondral bone, and formation of callus. At present, the etiology of osteoarthritis has not yet been clarified. On the surface of existing research, the occurrence of osteoarthritis is related to factors such as age, gender, family history, obesity, trauma and increased weight bearing of the joints. The incidence of osteoarthritis is positively correlated with age, and the study of its etiology and pathogenesis plays an important role in early diagnosis, termination of disease progression and effective treatment. Gene expression studies have been widely used to analyze DEGs and identify novel pathways. In this study, DEGs in 6-week PC compared with 17-week RC were identified based on gene expression profiling, among which 4821 upregulated genes and 4665 downregulated genes were observed.
Furthermore, functional enrichment analysis of the top 30 genes associate with chondrogenesis was performed to demonstrate the possible biological mechanisms. The top 10 pathways and associated biological processes were detected via the GO BP terms and KEGG pathway analyses. The majority of the top 30 upregulated and downregulated DEGs, including PTGS2, CCL20, CHI3L1, LIF, CXCL8, and CXCL12 were intensively enriched in immune-associated biological process terms, including inflammatory and immune responses. Moreover, ICAM1, COMP, and CNTN3 were involved in cell adhesion. In addition, the majority of the top 30 upregulated and downregulated DEGs were mainly enriched in NF-kappa B signaling pathway and TNF signaling pathway.
The PPI network of top 30 upregulated and downregulated DEGs was constructed. The results indicated that MMP1, MMP3, MMP13, and PTGS2 were hub genes. According to previous study, PTGS2, MMP1, MMP3, and MMP13 play a crucial role in the degeneration of articular cartilage, which accelerates the destruction of articular cartilage. MMP-1, also known as collagenase-1, is the first MMPs to be characterized and isolated from human fibroblasts. It is a fibroblastic collagenase that hydrolyzes collagen, gelatin such as I, II, III, VII, VIII, cell adhesive and aggrecan. It is abundantly expressed in OA cartilage and acts on newly synthesized type II collagen. In the case of compression or damage of chondrocytes, chondrocytes synthesize MMP-1 in a large amount, and the site of action is located at the Gly775-Leu776 site on the newly formed type II collagen alpha chain in the cartilage matrix, which is cleaved into 2. The A and B segments, which are 1/4 and 3/4 long, respectively, can be further decomposed by other MMPs. Type II collagen cleavage, destruction of collagen network structure, decomposition or loss of proteoglycans in ECM accelerated the degradation of cartilage matrix, destruction of cartilage and defects, leading to OA. Additionally, Freemont et al found that the expression sites of MMP1 and MMP3 in human knee OA cartilage were specific, and the abnormal expression period was not the same. MMP1 was mainly abnormal in the superficial layer of cartilage, and MMP3 was abnormally expressed in the deep layer of cartilage. MMP3 increased significantly in the early and late stages of OA. Moreover, MMP13, also known as collagenase 3, is mainly secreted by chondrocytes and is the most effective type II collagen degrading enzyme in the MMPs family. Its degradation ability is 5 to 10 times stronger than that of MMP1. MMP-13 can directly degrade the most abundant and characteristic type II collagen in ECM. Compared with MMP1, MMP13 plays a more significant role during cartilage degeneration. Although MMP1 and MMP13 cannot directly degrade proteoglycans, proteoglycans are mainly linked with type II collagen and hyaluronic acid in the form of polymers. MMP1 and MMP13 directly degrade type II collagen, which leads to the loss of the connection of proteoglycans and type II collagen. It further leads to the loss of proteoglycan, indirectly reduces the content of proteoglycan and causes the mechanical properties and deformation ability of cartilage to decrease, which accelerates the degeneration process of articular cartilage. Construction of PPI network of DEGs may be helpful for understanding the relationship of developmental chondrogenesis and OA. Additionally, cross-validation was performed to explore the relationship between the top 30 DEGs and genes from related studies. Among the top 30 upregulated and downregulated DEGs, there were 15 genes have been reported to be related to OA or developmental chondrogenesis. For instance, DKK1 levels correlate with osteoarthritis and are regulated by OA associated factors. CXCL12 levels are correlated with disease severity in patients with knee OA. Analysis of relationship between the top 30 DEGs and genes from related studies would be helpful for further study of OA and human developmental chondrogenesis.
In conclusion, the present study provides significant information that may aid in understanding the molecular mechanisms of developmental chondrogenesis. The top 30 upregulated and downregulated DEGs associated with developmental chondrogenesis were observed in this study, which may facilitate the research for cartilage tissue engineering and prevention of OA in the near future.
The authors would like to thank all co-investigators, and colleagues who made this study possible. The authors thank Dr Ayub Abdulle nur and Dr Chenxi Li for English language support in preparing manuscript.
Conceptualization: Jian Zhou, Wanchun Wang, Yingquan Luo.
Data curation: Jian Zhou, Xiadong Du, Wanchun Wang.
Formal analysis: Jian Zhou, Wanchun Wang.
Funding acquisition: Jian Zhou, Wanchun Wang.
Investigation: Jian Zhou, Yingquan Luo.
Methodology: Jian Zhou.
Software: Jian Zhou, Anqi Yu, Shuo Jie.
Validation: Jian Zhou, Anqi Yu, Xiadong Du, Tang Liu.
Writing – original draft: Jian Zhou, Wanchun Wang.
Writing – review & editing: Jian Zhou, Chenxi Li, Anqi Yu, Shuo Jie, Tang Liu, Wanchun Wang, Yingquan Luo.
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Keywords:Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.
bioinformatics analysis; enrichment analysis; human developmental chondrogenesis; PPI network module