Temporomandibular joint osteoarthritis (TMJOA) is an end-stage disease of temporomandibular joint disorder (TMD) characterized pathologically by synovial inflammation, cartilage degeneration, and subchondral bone remodeling. The clinical manifestations include chronic pain and masticatory difficulties that seriously affect a patient’s quality of life (Bush and Beier, 2013; Li et al., 2019). Although there are many studies on the etiology of TMJOA, its exact pathogenesis remains unclear (Zheng et al., 2018).

In recent years, studies on RNA sequencing have found that a variety of noncoding RNAs, including miRNA, lncRNA, and circRNA play an important role through competing endogenous RNA (ceRNA) networks in the development of osteoarthritis (OA) (Kostopoulou et al., 2015; Yin et al., 2017). CircRNAs and lncRNAs can act as endogenous miRNA molecular sponges and competitively inhibit miRNA regulation of target mRNAs (Memczak et al., 2013; Wang et al., 2016; Zheng et al., 2016; Wang et al., 2017; Xiong et al., 2018). Our previous study found that the expression of circRNAgcn1l1 was elevated in synovial TMJOA tissues and was able to regulate Tumor Necrosis Factor-alpha (TNF-a) secretion by synoviocytes through miRNA-330-3p, aggravating cartilage degeneration (Zhu et al., 2020). LncRNAs are another important group of miRNA sponges in addition to circRNAs and may be an important therapeutic target of TMJOA as an indispensable part of ceRNA regulation. For example, Fu et al. (2021) found that the lncRNA-h19/miR-124a/cdk2/mcp-1 axis could promote the proliferation of human rheumatoid arthritis (RA) synoviocytes and synovial inflammation, which may represent a new RA therapeutic strategy. Bi et al. (2019) concluded that inhibition of lncRNA picsar/miRNA-4701-5p could effectively inhibit the proliferation and invasion of synoviocytes as well as secretion of inflammatory factors in human RA. Thus, lncRNA picsar could be used as a new biomarker for RA. In addition, the lncRNA neat1/miRNA‑ 204‑5p/NF-κB axis and the lncRNA pint/miRNA-155-5p/socs1 axis have also been reported in human RA synovitis symptoms (Wang and Zhao, 2020; Xiao et al., 2021).

As there is a current lack of reports on lncRNA regulation in TMJOA synovitis, in this study, we performed high-throughput sequencing of lncRNAs, miRNAs, and mRNAs in human TMJOA synovial tissues. By constructing lncRNA-related ceRNA networks using bioinformatics analysis, we aimed to probe their potential role in the development of TMJOA.

LncRNAs are noncoding RNAs of more than 200 nucleotides in length (Xie et al., 2021). They can work as miRNA sponges to participate in regulating gene expression (Wang et al., 2019c; Wu et al., 2020). There is ample evidence that the relationship between lncRNAs and miRNAs plays an important role in OA progression (Xie et al., 2020). For example, Zhang et al. (2019) found that lncRNA malat1 could play a role in knee osteoarthritis by regulating chondrocyte proliferation, apoptosis, and extracellular matrix degradation through miR-150-5p/akt3. In another study, Liu et al. (2018) found that exosomes secreted by human stem cells contain lncRNA klf3-as1, which could regulate miR-206/git1, thereby affecting chondrocyte apoptosis. In addition, the lncRNA malat1/miR-146a-PI3K/Akt/mTOR axis and lncRNA foxd2-as1/miR-27a-3p/tlr4 axis have also been reported to regulate chondrocyte matrix degradation (Wang et al., 2019a; Li et al., 2020b). Some scholars have sequenced lncRNAs and analyzed differentially expressed lncRNAs in knee OA cartilage tissues, providing a basis for studying the regulatory role of lncRNAs in knee OA cartilage degeneration (Fu et al., 2015; Liu et al., 2016). As the initiating factor of OA, synovial inflammation can lead to the secretion of various inflammatory factors to promote cartilage degeneration, which is closely related to the severity and progression of OA (Kortekaas et al., 2016; Bhattaram and Chandrasekharan, 2017; van den Bosch, 2019). Many scholars have therefore also conducted many aspects of research on OA synovial inflammation. For example, Xiang et al. (2019) identified differentially expressed circRNAs in synovial tissues of knee OA by RNA sequencing, while further validation is however needed. This study previously identified differentially expressed circRNAs in TMJOA synovial tissues and found that circRNA_gcn1l1 was able to regulate TNF-a secretion in the synovium through miRNA-330-3p. Additionally, Xu et al. (2016) performed miRNA sequencing in TMJOA synovial tissues and found that miR-221-3p could regulate IL-1B and MMP1/9 secretion. However, none of the above studies involved the role of lncRNAs in OA and synovial inflammation symptoms. Studies on the relationship between lncRNAs and synovial inflammation have mostly come from RA. For example, Zou et al. (2018) sequenced lncRNAs in RA synovial tissue and found that lncRNA lerfs has a protective role in RA. Due to the current lack of reports of lncRNA regulation in TMJOA synovitis, we conducted lncRNA, miRNA, and mRNA sequencing in TMJOA synovial tissues in the hope of finding lncRNA/miRNA/mRNA axes that may play an important role in TMJOA.

The results of this study revealed that only one lncRNA molecule xist, was predicted to affect the expression of DE mRNAs through DE miRNAs.

LncRNA xist classically functions to silence the X chromosome to compensate for differences in the expression of X chromosome-associated genes between males and females (Czermiński and Lawrence, 2020). It can also play a role as an oncogene in some parenchymal tumors, including lung cancer and liver cancer (Yang et al., 2020). CeRNA network-related research is also mainly in the field of oncology. For instance, Li et al. (2020a) found that xist was significantly increased in both synovial and cartilage tissues of knee OA, which was consistent with the results of the analysis in this paper. They found that xist could help express osteopontin (OPN) in M1 macrophages in synovial tissues by binding miR-376c-5p, promoting macrophage cytotoxicity to chondrocytes, and then resulting in chondrocyte apoptosis. In another report, Liu et al. (2020) also confirmed that there was a significant upregulation of xist in knee OA cartilage and found that xist could facilitate DNA methyltransferase 3A (DNMT3A) expression and eventually exacerbate the destruction of chondrocytes by inhibiting miR-149-5p. Further, Wang et al. (2019b) reported that significant upregulation of xist was also confirmed in knee OA cartilage, and the xist/miR-1277-5p/mmp 13/adamts5 axis was found to have an important role in regulating OA disease development. Hence, xist can impact the course of OA through different targets and therefore may become an important target for the treatment of OA. Among lncRNAs, xist is a research hotspot with important functions. However, as most of the above studies were on cartilage tissue, the role of xist in synovitis is still unclear. Our in vitro experiments proved that blocking xist could protect synovial cells under inflammatory conditions.

Among the nine DE mRNAs predicted to be regulated by xist, ctsb was the hub gene selected by analysis, and mmp3 is considered an important cartilage matrix degradation marker. Besides, fos and grb2 products are molecules involved in the osteoclast differentiation pathway. The remaining five target genes are not involved in pathway enrichment or hub gene prediction. Thus ctsb, mmp3, fos, and grb2 were screened as subsequent research points in this work. The known functions of these four mRNAs in TMJOA have been summarized in Table 4. Our primary results showed that under inflammatory conditions, protein expression of CTSB, MMP3, c-FOS, and GRB2 decreases after inhibition of xist. This provided the evidence for our network construction.

Cathepsin B (CTSB), a lysosomal cysteine protease, is also a marker of the dedifferentiated phenotype of chondrocytes (Li et al., 2017). Research revealed that the DE miRNA hsa-miR-1271-5p could regulate CTSB. Zhang et al. (2021) documented that in chondrocytes, miR-140-5p could attenuate OA cartilage destruction by inhibiting CTSB/NLRP3. Ben-Aderet et al. (2015) found that CTSB was significantly higher in the synovial fluid in OA. However, the mechanism of CTSB in synovial tissue and synovitis is still unclear, and the analysis in this study yielded that the ceRNA network xist/miR-1271-5p/ctsb may function in synovitis.

Matrix metalloproteinase-3 (MMP3) is a classical molecule involved in cartilage destruction that is able to directly degrade the cartilage extracellular matrix and cause cartilage destruction by activating MMP9. MMP3 expression is directly related to the degree of inflammatory cell infiltration in the synovium. Further, increased metalloproteinase levels such as MMP3 are typical findings in OA (Berenbaum and Sellam, 2010). We found that the DE miRNA miR-365a-3p, a target of xist, was able to inhibit MMP3. It, therefore, constitutes the second ceRNA network, xist/miR-365a-3p/mmp3, in this study.

The results of GSEA and KEGG analysis both showed that the osteoclast differentiation pathway and osteoclast formation play an important role in the degeneration of subchondral bone (Zhu et al., 2019). This may be a bridge between synovitis and pathological remodeling of the subchondral bone. There are two core genes, fos and grb2 in the osteoclast differentiation pathway. The fos-encoded C-FOS protein is an important transcription factor in osteoclast formation, which is activated by the classical osteoclast formation regulator Receptor activator of nuclear factor kappa-B ligand (RANKL) and then activates the downstream NFATC1. Together with transcription factors such as MYC and NF-κB, C-FOS forms a complex network to function in promoting osteoclast formation. However, in this process, the regulation of C-FOS expression after activation by RANKL is not fully understood (Fujii et al., 2021). Therefore, whether C-FOS is also regulated by other molecules and thus affects its final expression needs to be confirmed. We found that the DE miRNA miR-199a-3p is able to bind fos, so it is worth exploring whether xist/miR-199a-3p can play a role in osteoclast formation through fos and whether it can be a therapeutic target for TMJOA.

GRB2 is a key factor in several tyrosine kinase signaling conversion pathways. Some studies have reported that GRB2 can promote osteoclast survival by activating Erk and can strengthen the adhesion of osteoclasts to the bone surface, leading to bone resorption (Levy-Apter et al., 2014). A recent study found that after the knockout of PIP5k1β kinase, GRB2 expression increased, and the ERK1/2 signaling pathway was more active, which promoted osteoclast formation (Zhao et al., 2020). Our preliminary in vitro experiments demonstrated that under inflammatory conditions, inhibition of GRB2 contributes to the decline of matrix degrading enzymes in synoviocytes. This provides a research basis for alleviating joint destruction by regulating GRB2.

Through the bioinformatics analysis above, four ceRNA regulatory axes were found in this study, including xist/miR-1271-5p/ctsb, xist/miR-365a-3p/mmp3, xist/miR-199a-3p/fos, and xist/miR-27b-3p/miR-1271-5p/grb2. These four networks may be involved in the development of synovial inflammation and are one of the important reasons why synovial inflammation can promote cartilage and subchondral bone degeneration. Their roles still need to be confirmed by further in vitro and in vivo studies. Thus, in our future work, we will focus on the validation of the aforementioned ceRNA networks.