As a crucial part of the tibiofemoral articulation and an articular cartilage of the knee joint, the meniscus protects the underlying articular cartilage, contributing to normal function of the knee joint and protecting the articular surfaces (Noble and Turner, 1986; Huang et al., 2019). Meniscus injuries include traumatic tears caused by sports injury and age-related degenerative tears. Degeneration of the meniscus further increases the risk of traumatic tears. In recent years the incidence of meniscus injury is on rise probably due to the general aging of the population. Aging is a time-dependent decline of physiological abilities. Physiological degeneration is a major risk factor for age-related diseases such as type 2 diabetes, cancer, and dementia (Degirmenci and Lei, 2016). Many changes occur in articular cartilages with aging, including dysregulated expression of related genes, deterioration of mechanical properties and degenerative changes in the morphology (Sacitharan and Vincent, 2016). Age-related tissue changes, such as myxoid degeneration, collagen fiber thickening, and accumulation of advanced glycation end products, ultimately increases the tissue vulnerability of the meniscus (Pauli et al., 2011). Knee cartilage thickness is also negatively correlated with age. All these changes aggravate the course of osteoarthritis, increase the incidence of meniscal tears in the elderly population, and seriously affect people’s quality of life. However, there are few studies on meniscus aging, and key genes involved in meniscal aging are still remaining to be identified.

Long non-coding RNAs (lncRNAs), which do not have protein-coding functions, have received increasing research attention in recent years (Quinn and Chang, 2016). LncRNAs play key roles in many biological processes. Studies have shown that lncRNAs play important roles in the aging process of tissues and organs. For example, the expression of lncRNA H19 is downregulated during endothelial cell senescence. LncRNA H19 controls senescence, angiogenic sprouting, proliferation, and inflammatory activation of endothelial cell via inhibiting STAT3 activation (Hofmann et al., 2019). Su et al. found 88 significantly upregulated lncRNAs and 46 significantly downregulated lncRNAs in the cochlea of aged C57BL/6 mice. LncRNA AW112010 alleviated age-related hearing loss by promoting mitochondrial biogenesis to maintain mitochondrial function and hair cell survival in cochlear hair cells (Su et al., 2019). Moreover, dysregulated lncRNAs were involved in meniscus degeneration. Interleukin-1 β (IL-1β) treatment of degenerative meniscus resulted in significant alterations in 375 mRNAs, 15 miRNAs, 56 lncRNAs and 90 circRNAs. LncRNA LOC107986251 is an important player in meniscal degeneration (Jiang et al., 2021). Zhao et al. performed an analysis on the GSE98918 dataset, which included 24 meniscus samples and related clinical data. The expressions of 208 lncRNAs and mRNAs were significantly differential between patients with knee osteoarthritis and non-knee osteoarthritis group. In the knee osteoarthritis group, a total of five lncRNAs were significantly associated with meniscus degeneration (Zhao et al., 2020a). However, until now, the role of lncRNAs in age-related change of meniscus is poorly known.

In order to investigate the molecular mechanisms of age-related change of meniscus and further explore potential biologic therapeutic targets, we performed microarray analysis of meniscus to determine lncRNAs and mRNAs expression profiles in young and aging adults. Then, we carried out bioinformatics analysis of differentially expressed molecules to identify key molecules and functional pathways in the aged meniscus tissues.

Age-related degeneration of the meniscus is a pathological process that occurs at the tissue, cell, and molecular level of the meniscus. It is manifested as significant morphological and physiological changes of the meniscus, and finally manifested as a decrease in its load-bearing capacity. Since aging is an inevitable process, finding mechanisms to protect the meniscus tissue from avoidable damage has become the focus of research. In order to understand the influence of age factors on meniscus tissues, we analyzed the changes of lncRNA and mRNA expression profiles in meniscus tissues of young and aging people by microarray analysis. A total of 1,608 lncRNAs and 1809 mRNAs have undergone significant changes.

We first analyzed the function of DE mRNA. After verification by qRT-RCR, we found the expression of metalloproteinase-2 (MMP-2) and type I α 2 the expression of colagen (COL1A2) were significantly decreased in the aging group. ECM of meniscus plays a key role in regulating cell activity due to non-vascular nature of the adult meniscus. The ECM is composed of various macromolecules, mainly including types I and III collagens, as well as degradative components such as matrix MMPs (Fourniere et al., 2021). Meniscus ECM plays an important functional role in regulation of the physicochemicall and mechanical environments at multiple scales, which affects overall joint health, cartilage homeostasis, and chondrocyte metabolism (Wilusz et al., 2014). MMPs degrades structural components of ECM, such as collagen (Amaral et al., 2020). MMPs are directly related to almost every biological process that involves ECM remodeling. In the aging process, changes in collagen structure and disorder of MMP will affect ECM remodeling, leading to a variety of diseases, such as vascular diseases, rheumatoid arthritis, osteoarthritis, fibrosis and cancer (Panwar et al., 2018). In many animal models and human diseases, imbalances in MMP activity are associated with myocardial aging status, even in the absence of cardiovascular disease (Meschiari et al., 2017). In aging mouse myocardium and cartilage, increased MMP activity is associated with increased inflammation, ECM deposition, and decreased angiogenesis (Meschiari et al., 2017; Sebastian et al., 2020). Dysregulated ECM leads to myocardial fibrosis and cartilage ECM degradation, which results in dysfunction at the cellular, extracellular, and whole organ levels. MMP-mediated ECM degradation is the core process of intervertebral disc degeneration. In nucleus pulposus cells, miR-874-3p targets and inhibits the expression of MMP2 and MMP3(Song et al., 2021), and PART1 controls nucleus pulposus cells degeneration through the miR-93/MMP2 pathway (Gao et al., 2020). The meniscus is a fibrocartilage and contains a lot of type I collagen. Type I collagen is a heterotrimer protein consisting of one α1 chains (COL1A1) and two α2 chain (COL1A2) (Karsenty and Park, 1995). COL1A2 is a major component of ECM. Some studies have reported that COL1A2 is involved in regulation of the osteoarthritis, Ehlers-Danlos syndrome, osteogenesis imperfecta (Wu et al., 2019). Our research showed that the mRNA expression of MMP2 and COL1A2 significantly decreased in the aging group. We believed that abnormal expression of MMP2 and COL1A2 in the aging group may contribute to ECM disorder. This might be one of the reasons that affect the structural and mechanical integrity of the meniscus.

During aging, the level of apoptosis has changed in many tissues (Tower, 2015). For example, excessive proliferation and decreased apoptosis of vascular smooth muscle cells in aged humans and mice may contribute to plaque formation (Qian et al., 2011). However, in other tissues, the increase in apoptosis leads to a decrease in the number of tissue cells, and the loss of cells increases the destruction of tissue homeostasis and dysfunction. The process of intervertebral disc degeneration is associated with higher rates of apoptosis and senescence. This results in cell loss in the nucleus pulposus tissue led to significant changes in the disc morphology and physiology which ultimately resulting in its decreased capability to bear compressive loads (Cazzanelli and Wuertz-Kozak, 2020). In our research, we found that the 18 DE mRNAs identified in the three databases are associated with cartilage injury and are mainly involved in the Hippo signaling pathway. The Hippo signaling pathway is a highly conserved pathway involved in tissue development and regeneration, which controls organ size by regulating cell proliferation and apoptosis (Ma et al., 2019; Meng et al., 2021). Activation of Hippo signaling pathway by overexpression of mammalian Ste-20 like kinase 1 (Mst1) or downregulation of YAP inhibits the development of Natural Killer T-Cell Lymphoma in nude mice xenograft models (Chang et al., 2019). During ischaemia-reperfusion injury or myocardial infarction, the Hippo-YAP pathway is activated and then activates Mst1 to increase caspase activation and cardiomyocyte apoptosis. Since the human heart lacks the ability to self-repair, loss of cardiomyocytes after injury such as myocardial infarction can lead to heart failure and death (Wang et al., 2018). Our results implicated that the Hippo pathway is involve in the aged meniscus. The Hippo pathway might be involved in meniscal tissue cell loss through apoptosis, leading to meniscal morphological and physiological changes. Currently, the Hippo pathway has emerged as a potential therapeutic target for controlling pathological cardiac remodelling. For example, the new inhibitor XMU-MP-1 can be used to pharmacologically inhibit Mst1/2, the core component of the Hippo pathway, to control the adverse effects of hypertrophy caused by pressure overload (Triastuti et al., 2019). Therefore, amelioration of meniscal degeneration by controlling the Hippo pathway may become a new research direction that deserves further investigation.

In addition to ECM degradation and cell loss, inflammation also plays an important role in the degenerative process. The 18 DE mRNAs that we identified in the three databases were involved in TGF-beta signaling pathway. It was suggested that inflammation occurred during meniscus degeneration. However, the regulatory mechanism of TGF-beta signaling pathway during aging is still unclear. In recent years, studies have found that a large number of lncRNAs play a regulatory role in inflammatory responses, including TGF-beta signaling pathway (Hodgson et al., 2019). Tang et al. (2019) found that lncRNA-ATB promotes TGF-β induced glioma cell invasion through NF-κB and P38/MAPK pathways. LncRNA H19 expression was downregulated in vitreous humour of patients with proliferative diabetic retinopathy. LncRNA H19 via TGF-β Influences endothelial mesenchymal transition during diabetic retinopathy (Thomas et al., 2019). LncRNA SNHG3 promotes proliferation and migration of non-small-cell lung cancer cell by activating TGF-β pathway and IL-6/JAK2/STAT3 pathway (Shi et al., 2020). Through CNC analysis of DE lncRNAs, we found that lncRNA AP001011 1, which is involved in modulating inflammation, Inhibits the expression of tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), and AC124312.5 upregulates the expression of TNFRSF11B. TNFRSF11B is a member of tumor necrosis factor receptor super family (TNFRSF). It is a secreted for tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) (Luan et al., 2020). The expression of TNFRSF11B is significantly upregulated in patients with osteoarthritis. The miR-145 plays an important role in chondrocyte proliferation and fibrosis by directly targeting TNFRSF11B, which inhibits the proliferation and fibrosis of OA (Wang et al., 2017). Upregulation of TNFRSF11B expression in injured osteoarthritic cartilage is a factor driving the transition of chondrocytes into osteoblasts in 3D vitro chondrogenesis mode (Ruiz et al., 2021). TNFRSF11B gene encodes osteoprotegerin. Due to the important role of osteoprotegerin in bone biology, TNFRSF11B gene is considered as a candidate gene for osteoporosis. Some studies believe that TNFRSF11B gene t245g (rs3134069) polymorphism is a risk factor for osteoporotic fracture (Boronova et al., 2015). However, there is still a lack of research on the role of TNFRSF11B in aged meniscus, especially its regulatory mechanism. Through CNC analysis, we found that AP001011 inhibited the expression of TNFRSF11B and AC1243125 upregulated the expression of TNFRSF11B, which provided a direction for further study on the mechanism of regulating TNFRSF11B during aging.

Our CNC analysis also showed that lncRNA human leukocyte antigen complex group 11 (lncRNA HCG11) upregulated the expression of Smd5. It has been confirmed that lncRNA HCG11 is involved in regulating the progression and prognosis of various diseases. HCG11 inhibits growth and invasion of cervical cancer cell by sponging miR-942-5p and targeting growth factor-independent transcription repressor 1 (Zhang et al., 2020). Moreover, HCG11 promotes the expression of MMP13 and exacerbates osteosarcoma through sponging miR-579. However, the relationship between HCG11 and meniscus degradation has not been studied. According to our research, we proposed that HCG11 expression is significantly upregulated in the meniscus tissues in aging group, and Smad5 may be the target molecule of HCG11.

LncRNA act as miRNA sponges alleviating the inhibitory effects of miRNAs on target genes and increasing target gene amounts, known as ceRNA. Our ceRNA analysis indicated that lncRNA POC1B-AS1 regulates the expression of bone morphogenetic protein 2 (BMP2), MAPK14, Smad3 and TGF-beta receptor 2 (TGFBR2) through sponging miRNA. BMP2 is a superfamily member of TGF-β, it is one of the main chondrogenic growth factors involved in cartilage regeneration (Balaskas et al., 2020). Subcutaneous stem cell implantation studies have shown that BMP2 induces cartilage formation but also promotes endochondral ossification during ectopic bone/cartilage formation (Zhou et al., 2016). Melatonin improves age-related bone loss and osteoporosis by upregulating the expression of BMP2. Our findings suggested that lncRNA POC1B-AS1 regulates the expression of BMP2 by sponging miR130a-3p, miR136-5p, mir18a-3p, and miR608. However, the involvement of these miRNAs in meniscus degradation should be studied further.

In this work, we also predict the m⁶A modification site and the subcellular locations of 4 DE lncRNAs. The m⁶A modification is the most abundant internal modification in mRNAs and occurs also in lncRNAs (Zhao et al., 2017). Alterations in RNA m⁶A methylation status can lead to cell dysfunction and disease. At present, m⁶A-modified lncRNAs have received extensive attention. M⁶A modification may control gene expression by regulating the translation efficiency and stability of lncRNAs (Coker et al., 2019). Shen et al. (2021) found that decreased m⁶A levels of lncRNA-XR_343955 in rat aortic tissue affect the inflammatory response through the cell adhesion molecule pathway during lipopolysaccharide-induced sepsis. The decrease in m⁶A modification of lncRNA XR_346,771 may be related to cation import in rat cardiac tissue during sepsis (Han et al., 2021). Moreover, the function of lncRNA is closely related to their subcellular localization. In the nucleus, lncRNAs regulate gene expression at the epigenetic and transcriptional levels, and in the cytoplasm, they regulate gene expression at the post-transcriptional and translational levels (Zhao et al., 2020b). Our results showed that lncRNA HCG1 was located in nucleus and lncRNA POCIB-AS1 was located in cytoplasm. This indicated that the lncRNA HCG1 and lncRNA POCIB-AS1 have different functions. M⁶A modification site and the subcellular locations analysis provided a new direction for in-depth study of these lncRNAs.

In summary, this study investigated the lncRNAs and mRNAs expression profile of meniscus between young and aging group, and further analyzed regulatory relationships between lncRNAs and mRNAs that may be associated with change of aged meniscus. ECM degeneration, apoptosis, and inflammation were the focus of this study, especially the abnormal expression of lncRNAs and its regulatory role. At present, people have a high demand for new and targeted therapies methods that can delay the degeneration of the meniscus and reduce the risk of meniscus tear. Several possible approaches are currently being studied and tested. Among them, cell therapy, endogenous repair strategies, and treatments based on biological factors such as no coding RNA (miRNA, lncRNA et al.) are the most promising. In order to prevent or limit the impact of meniscal degeneration on articulation, we analyzed the molecular background of meniscus in the aging people used bioinformatics methods, providing directions for the development of new targeted therapies.