With the in-depth study of the gene regulation network related to the pathogenesis of OA, the regulatory role of non-coding ribonucleic acid (RNA) has been continuously revealed. And it has been found that a large number of lncRNAs play an important role in the regulation of osteoarticular cartilage degeneration(8–10) lncRNA can compete with endogenous RNA (ceRNA), miRNA sponge, to play a regulatory role(6) lncRNA participates in gene regulation as guide, signal, bait and scaffold. The specific regulation mechanism is mainly divided into four aspects, which regulates the degeneration of articular cartilage by regulating transcription factors, transcription process, mediating post-transcriptional regulation of miRNA and mRNA, and regulation of nuclear structure(19) At present, the most concerned research of lncRNA is that it has a "sponge" endogenous competition effect with miRNA that has the same binding site as itself, inhibiting the regulation of miRNA on mRNA, thereby affecting protein expression. The more binding sites there are, the stronger the "sponge effect" and the more obvious the inhibitory effect of lncRNA on miRNA. Under normal circumstances, different RNAs (such as lncRNA, miRNA, and mRNA) maintain a balanced state and when an RNA is abnormally expressed, the balance breaks and causes disease(20) Non-coding RNA with a common response element (microRNA response elements, MREs) can compete with mRNA endogenously to bind miRNA and inhibit miRNA's negative regulation of mRNA. Similarly, reducing ceRNA levels can lead to up-regulation of the target gene expression, which may ultimately affect cellular biological processes(21)
The expression differences of in lncRNA and miRNA in OA chondrocytes cultured with SDF-1 and those without SDF-1 were analyzed by high-throughput sequencing. A total of 52,741 lncRNAs were found to be changed in expression. Through analysis, a total of 186 lncRNA changes were found to have significant statistical differences, of which 88 lncRNAs were up-regulated and 98 lncRNAs were down-regulated. A total of 119,205 miRNAs were found to have differential expression and a total of 684 miRNAs had significant statistical differences in their expression, of which 323 miRNAs were up-regulated, and 361 miRNAs are down-regulated. Subsequently, through GO enrichment and KEGG enrichment analysis, we analyzed the function of differential mRNAs and summarized the characteristics of differential lncRNAs. A series of key genes have been identified, which may be the pathological mechanism or biomarker of chondrocyte degeneration. Through GO analysis, we found that the signaling pathways of miRNA and its target genes are enriched in receptor regulation activities (molecular functions); secondary lysosomes (cell composition); signaling pathways regulatory by lipopolysaccharide (biological processes), type Ⅰ interferon signaling pathways, regulation of ionic transmembrane transporter activity.
Receptor regulatory factors such as Toll-like receptors (TLRs) are evolutionarily conserved molecules that promote immune responses by recognizing molecular patterns related to microorganism. During infection, TLR signaling is necessary for the proper activation of the body's immune response(22) TLRs produce a large amount of IL-1β and TNF-α inflammatory factors by activating the NK-κB inflammatory signaling pathway. Liu et al(23) found that the expression of TLR-2, NF-κB, MMP-13 and related inflammatory factors were significantly up-regulated with the severity of OA lesions, suggesting that TLR-2 / NF-κB signaling pathway may be involved in the occurrence of OA.
Lysosome is a complex intracellular organelle that positively interacts and involves in phagocytosis, autophagy, exocytosis, receptor circulation and regulation, intracellular signaling, immunity, chromatolysis, and bone biology. The accumulation of lysosomal storage materials not only changes its function, but also affects the function of the entire cell. Cells can digest proteins and organelles through phagocytosis by lysosomes. When intra-articular hemorrhage occurs, lysosomes release degrading enzymes and sugar Decreased protein (PG) concentration reduces chondrocyte synthesis activity and aggravates articular cartilage degeneration(24) The inducer SDF-1 used in this research is a degrading enzyme and a high concentration of SDF-1 can interact with the CXCR4 receptor on the surface of chondrocytes and accelerate the degradation of Ⅱ-collagen through the up-regulation of MMPS, leading to cartilage degeneration(25) Chondrocytes are non-excited cells. The multiple ion channels present on the cell membrane are the material basis for the cell to carry out various life activities, including transporting ions necessary for cell metabolism, regulating osmotic pressure inside and outside the cell, participating in the formation of electrical impulses, participate in signal transmission to adapt organisms to environmental conditions(26, 27)
Pathway analysis showed that miRNA and its target genes were enriched in cytokine-cytokine receptor interaction, osteoclast differentiation, NF-κB signaling pathway, TGF-b signaling pathway, and Ca2+ signaling pathway. Cytokines regulate the balance of anabolic and catabolic metabolism of cartilage matrix. And cytokines are divided into decomposing cytokines and synthetic cytokines according to the characteristics of cytokines regulating metabolism. The balance and imbalance between them are root causes of the degradation and destruction of the cartilage matrix in osteoarthritis. A large number of cytokines are involved in this pathway, such as tumor necrosis factorα (TNF-α), interleukin-1 (IL-1), IL-6, IL-2, and interferon-γ (IFN-γ). These cytokines penetrate into the synovium to produce an inflammatory response. Besides, these cytokines can activate synovial cells and stimulate the release of MMPs into the synovial fluid, leading to cartilage degradation(28, 29)
Currently, the most studied cytokines that promote chondrocyte catabolism are IL-1 and TNF-α. IL-1 not only inhibits the synthesis of characteristic matrix components type II collagen and aggrecan by articular chondrocytes, but also stimulates articular chondrocytes to secrete protease that degrades cartilage matrix components, induces articular chondrocytes to express type Ⅰ and type II collagen degradation, and promote the degeneration of articular chondrocytes(30) TNF-α also plays an important role in OA cartilage degeneration. The mechanism of action of TNF-α is similar to IL-1, including promoting the generation of MMP and inhibiting the synthesis of cartilage matrix, etc. Studies have shown that TNF inhibits the expression of type Ⅱ collagen and connexin genes through the pathway of MEK1 / 2 and nuclear factor (NF) -κB, which in turn interferes with the synthesis and reconstruction of articular cartilage(31) As a transcription factor regulating gene expression, studies have found that the NF-kB signaling pathway is activated in OA occurred articular cartilage and synovial cells(32) NF-kB regulates the response to joint injury and inflammation by regulating the cytokines IL-1β, TNF-α, etc.(33–37)
In addition, the PPI network revealed 10 genes that have a higher possibility in association with the pathological process of chondrocyte degeneration: CXCL10, ISG15, MYC, MX1, OASL, FIICT1, RSAD2, MX2, IFI44LBST2. Chemokines are a class of small molecule peptides with chemotactic effects that can attract immune cells to produce an immune response locally. Chemokines, mainly CXC and CC, and their receptors are expressed in human chondrocytes and their corresponding receptors while expression increased in OA articular cartilage(38) Chemokines are involved in cartilage destruction by inducing some related enzymes, mainly N-acetyl-β-D-glucosidase (NAG) and MMP. NAG is the main lysosomal glycosidase in OA synovial fluid and has the function of catalyzing the hydrolysis of glucosamine polysaccharides, and then causes the destruction of cartilage.
With the occurrence of OA, the cartilage surface is activated by a variety of chemokines, releasing enzymes that mediate the destruction of the cartilage matrix(39, 40) Kostopoulou et al(41) and Tardif et al(42) found that OA-related miRNA can inhibit matrix metalloproteinase 13 (MMP-13). MMP-13 is a class of human proteases with important biological functions in OA. It can degrade cartilage extracellular matrix, destroy articular cartilage, and cause and aggravate OA.
The human c-Myc gene is located on the eighth pair of chromosomes and consists of three exons. Its expression product is a protein containing 439 amino acids, which exists in the nucleus. c-Myc is not expressed in normal chondrocyte nucleus, but scattered in apoptotic chondrocyte nucleus. In the comparison of articular chondrocytes and OA articular chondrocytes in normal people, it was found that the degree of OA articular chondrocyte apoptosis was positively correlated with the degree of cartilage degeneration, and c-myc participated in the whole process of chondrocyte apoptosis. The mechanism of c-Myc causing apoptosis may be due to the imbalance of normal cell cycle, which leads to the inhibition of cell growth.(43)