CAMP Gene Promoter Methylation Induces Chondrocyte Apoptosis By Inhibiting ROS Levels And Inammatory Response

Objective: The occurrence of osteoarthritis is related to genetic and environmental factors. Among them, the change of chondrocyte gene expression pattern regulated by epigenetic modication is an important participant. This study analyzed the effect of CAMP gene methylation on the level of oxidative stress and inammation of chondrocytes. Methods: We analyzed the changes of the transcriptome in the articular cartilage tissue of osteoarthritis patients (OA) from the GSE117999 dataset. The GSE48422 dataset was used to analyze the changes in the methylation level of osteoarthritis cells. MTT assay and ow cytometry analysis of short hairpin RNA (shRNA) silencing CAMP gene and 5μM 5-Aza-2’-Deoxycytidine (AZA) treatment on the proliferation and apoptosis of Human Chondrocytes Osteoarthritis (HC-OA) cells. The DCFH-DA assay was used to detect the level of reactive oxygen species (ROS), and the expression level of inammatory factors was analyzed by Western Blot. Results: The expression of CAMP in cartilage tissue of OA patients was up-regulated, and the level of methylation was down-regulated. CAMP was highly expressed in osteoarthritis articular cartilage cells. Silencing CAMP inhibited the proliferation of HC-OA cells and promoted their apoptosis. CAMP gene methylation inhibited ROS levels and TNF-α expression levels in HC-OA cells, and promoted TGF-β expression. CAMP gene methylation inhibited the proliferation of HC-OA cells and promoted their apoptosis. Conclusion: CAMP gene promoter methylation induces chondrocyte apoptosis by inhibiting ROS levels and inammation.


Introduction
Osteoarthritis (OA) is a common age-related degenerative disease. Its main pathological feature is articular cartilage degeneration, which is mainly manifested by the loss of articular cartilage, the formation of osteophytes, subchondral bone sclerosis and synovium in ammation, etc., clinical symptoms such as pain, joint deformity and dysfunction may appear in the later clinical stage [1][2][3].
Research in recent years has shown that epigenetic modi cation plays an important role in the pathogenesis of OA [4,5]. Epigenetic modi cation mainly includes DNA methylation, histone modi cation, chromatin remodeling and non-coding RNA, among which DNA methylation is closely related to in ammatory diseases [6,7]. At present, DNA methylation studies of OA mainly focus on articular cartilage, because articular cartilage is the core tissue involved in the disease process [8]. In addition, DNA methylation has become an important regulator of chondrocyte dedifferentiation, severely destroying the results of autologous chondrocyte implantation in patients with cartilage injury [8]. Therefore, it is of great signi cance to study the DNA methylation pro le of chondrocyte dedifferentiation.
At present, the methylation sites or regions identi ed in OA genome-wide methylation studies are mostly enriched in genes related to immunity or in ammation, cartilage development, transcription factor regulation, and protease activity [9,10]. Although OA was once considered a non-in ammatory disease, it has been con rmed that in ammation is very important in the occurrence of OA [11].
Tumor necrosis factor α (TNF-α) is one of the main pro-in ammatory cytokines related to the pathogenesis of OA, it can stimulate the release of matrix metalloenase-1 (MMP-1), MMP-3 and MMP-13 in OA patients, and inhibit the synthesis of proteoglycans and type II collagen [12]. Transforming growth factor β (TGF-β) is a member of the TGF-β superfamily. It transmits signals to the nucleus through the TGF-β receptor and its intracellular signaling system to play a variety of different physiological regulatory roles [13,14]. Studies have shown that TGF-β plays an important role in maintaining normal articular cartilage and joint repair [15].
Some researchers have used the demethylating agent AZA for in vitro intervention of OA chondrocytes.
For example, Iliopoulos et al. [16] found that AZA could reduce the methylation level of the leptin gene promoter of chondrocytes and increased the expression of leptin mRNA, which then activated matrix metallopeptidase 13 (MMP-13). Kim et al. [17] reported that when OA articular chondrocytes were cocultured with 10 µM AZA for 8 days, the methylation level of 6 CpG islands in the SOX-9 promoter region of chondrocytes decreased and the expression level of SOX-9 increased.
The CAMP gene (also known as LL-37) is located on chromosome 3p21.31. The CAMP protein is composed of 37 amino acids in a spiral shape and has a broad-spectrum antibacterial activity. Studies have shown that CAMP has defensive effects, such as regulating in ammation, inducing immune cells to injured or infected sites, binding and neutralizing Lipopolysaccharide (LPS), promoting epithelialization and repairing the damage [18]. Some researchers had found that CAMP affected the expression and distribution of TLR in tissue mast cells [19]. CAMP can be used as a powerful inducer of CCL3 and ROS generation [19]. Research by Li et al. [20] showed that CAMP could promote epithelial and smooth musclelike differentiation of adipose stem cells through Wnt/β-Catenin and NF-κB pathways.
In this study, we analyzed CAMP expression and methylation levels in OA chondrocytes through the GSE117999 dataset and GSE48422 dataset, and analyzed the effects of CAMP methylation on chondrocyte oxidative stress and in ammation level in vitro, as well as the impact on chondrocyte proliferation and apoptosis. It is of great signi cance for further elucidating the mechanism of OA, and it also provides a basis for early diagnosis and treatment of OA.

Datasets
Both the GSE117999 dataset and GSE48422 dataset [21] can be obtained from the Gene Expression

Cell transfection and 5-Aza-2'-Deoxycytidine (AZA) treatment
In order to study the effect of CAMP on the proliferation and apoptosis of chondrocytes, we used small hairpin RNA to silence CAMP gene expression (sh-CAMP), no template control(sh-NC) and no transfection group (Control) as controls. According to the manufacturer's instructions, HC-OA cells (5 × 105 cells/well) were transfected with Lipofectamine 3000 (Life Technologies, Gaithersburg, MD, USA) and incubated in a medium containing 10% FBS for 48 hours. The detailed process of AZA processing has been previously reported [22]. Quantitative reverse transcription-PCR (qRT-PCR) and Western blot were used to detect transfection e ciency.    Fig. 1a). The analysis of the GSE48422 dataset [21] showed that the level of CAMP methylation was down-regulated ( Table 2, Fig. 1b).

CAMP was highly expressed in osteoarthritis articular cartilage cells
We analyzed the expression levels of CAMP in the cartilage tissue of 8 cases of OA patients undergoing total knee arthroplasty and 5 cases of trauma requiring high amputation (excluding OA) (Control). The results showed that the expression levels of CAMP mRNA and CAMP protein in the articular cartilage tissue of KOA patients were signi cantly higher than those of the Control group (p = 0.002, p < 0.0001, Fig. 2a, 2b). Our analysis in Human Chondrocytes Osteoarthritis (HC-OA) cells and Human Chondrocytesarticular (HC-A) cells showed that the CAMP mRNA level and CAMP protein level in HC-OA cells were signi cantly higher than those in HC-A cells (p < 0.001, p < 0.0001, Fig. 2c, 2d).

Silencing CAMP gene inhibited the proliferation of osteoarthritis articular cartilage cells and promoted their apoptosis
In order to study the role of CAMP gene in the proliferation and apoptosis of chondrocytes in OA patients, we used small hairpin RNA to silence CAMP gene (sh-CAMP), no template control (sh-NC) and no transfection group (Control) as control. QRT-PCR and Western Blot results con rmed that CAMP gene was successfully knocked out in HC-OA cells (p < 0.001, p < 0.01; Fig. 3a, 3b). MTT assay results showed that compared with the Control, the proliferation ability of HC-OA cells was signi cantly inhibited after sh-CAMP transfection (p < 0.01, Fig. 3c). Flow cytometry detection of cell apoptosis showed that the apoptotic rate of HC-OA cells after sh-CAMP transfection was signi cantly higher than that of Control (p < 0.01, Fig. 3d).

CAMP gene methylation inhibited ROS levels and in ammatory response levels in osteoarthritis articular cartilage cells
In order to further study the effect of CAMP methylation on the level of oxidative stress and in ammatory response levels in HC-OA cells, we set up 3 groups, namely Control, sh-CAMP transfected HC-OA cell group (sh-CAMP), 5 µM 5-Aza-2'-Deoxycytidine (AZA) treatment group. First, Western Blot results showed that compared with the Control, the CAMP protein expression in HC-OA cells was signi cantly downregulated after sh-CAMP transfection, and CAMP expression in HC-OA cells was signi cantly increased after AZA treatment (Fig. 4a). After transfection with sh-CAMP, the level of ROS in HC-OA cells decreased signi cantly, while the level of ROS in HC-OA cells increased after AZA treatment (Fig. 4b). Western Blot results showed that compared with Control, TNF-α levels in HC-OA cells were signi cantly decreased after sh-CAMP transfection, and TNF-α levels were signi cantly increased after AZA treatment (Fig. 4c). The level of TGF-β in HC-OA cells was signi cantly increased after sh-CAMP transfection, and AZA treatment down-regulated the expression of TGF-β (Fig. 4d). These results showed that CAMP gene methylation down-regulated the expression of CAMP in HC-OA cells and inhibited the level of oxidative stress and in ammatory response levels.

CAMP gene methylation inhibited the proliferation of osteoarthritis articular cartilage cells and promoted their apoptosis
In order to further study the effect of CAMP gene methylation on the proliferation and apoptosis of HC-OA cells, we set up 3 groups, namely Control, sh-CAMP, and AZA. Western Blot analysis showed that, compared with Control, the CAMP protein in the sh-CAMP transfection group was signi cantly downregulated, and the CAMP protein expression level was signi cantly increased after AZA treatment (Fig. 5a). MTT assay results showed that compared with Control, the proliferation ability of HC-OA cells in the sh-CAMP transfection group was inhibited, and the proliferation ability of HC-OA cells was signi cantly increased after AZA treatment (Fig. 5b). The results of ow cytometry showed that compared with Control, the apoptotic rate of HC-OA cells in the sh-CAMP transfection group was signi cantly increased, and the apoptotic rate of HC-OA cells was signi cantly decreased after AZA treatment (Fig. 5c). These results showed that CAMP gene methylation inhibited the proliferation of osteoarthritis articular cartilage cells and promoted their apoptosis.

Discussion
In this study, we extracted and analyzed the differentially expressed genes and methylation levels in chondrocytes from public databases, and found that CAMP (also known as LL-37) was up-regulated in the articular cartilage of patients with OA, the methylation level of CAMP gene was down-regulated. We found that CAMP protein expression was up-regulated in articular cartilage tissue of OA patients and HC-OA cells. After silencing the expression of the CAMP gene, we found that the proliferation of HC-OA cells was inhibited, accompanied by a signi cant increase in the apoptotic rate. After demethylation, the level of ROS and in ammation in HC-OA cells increased signi cantly, the cell proliferation ability was signi cantly enhanced, and the apoptosis rate was signi cantly reduced. Therefore, we speculated that CAMP gene promoter methylation induced chondrocyte apoptosis by inhibiting ROS levels and in ammatory response levels.
Current research evidence shows that risk factors for OA include genetics, in ammation, environmental factors, age, stress stimulation, etc. [23,24]. In recent years, more and more studies have found that epigenetic modi cation plays an important role in the pathogenesis of OA. Epigenetic modi cation works by regulating gene transcription or post-transcriptional regulation, including DNA methylation, histone modi cation, chromosome remodeling and non-coding RNAs (ncRNAs) [6,25,26]. Usually DNA methylation occurs in cytosine, guanine, and nucleotides (CpG), which is mainly manifested as the phenomenon of conversion of cytosine to 5-methylcytosine [27,28]. The research on DNA methylation function mainly focuses on the transcription start site of genes [29]. Methylation of the promoter region can inhibit gene expression, on the one hand, methylation of the CpG site directly interferes with the binding of transcription factors to DNA in the regulatory region; on the other hand, methylated DNA and methylated CpG binding region proteins such as MeCP2 speci cally bind to form a complex, which restricts the passage of transcription factors to their binding sites, thereby inhibiting gene expression [30][31][32].
In this study, we found that the expression level of CAMP protein increased signi cantly after AZA treatment. The reason might be that the methylation level of the promoter region of CAMP gene decreased after AZA treatment, and the binding e ciency of CAMP gene transcription factors increased, as a result, the expression level of CAMP increased signi cantly. At present, researchers have tried to use AZA for in vitro intervention of OA chondrocytes. For example, Alvarez-Garcia et al. [33] treated TC28 cells with AZA and the expression of ATOH8 and TBX4 increased signi cantly. In addition, some researchers found that the DNA methylation level of the iNOS enhancer-5.8 kb CpG site decreased after AZA treatment, and the iNOS expression level increased, the methylation of the iNOS enhancer could inhibit the cell cycle process by downregulating NF-κB level and reduce the pro-in ammatory response, which had important therapeutic signi cance for OA [34].
In this study, we found that CAMP was highly expressed in articular cartilage cells of OA patients. Silencing the expression of CAMP gene inhibited the proliferation of osteoarthritis articular cartilage cells and promoted their apoptosis, suggesting that CAMP may play an important role in the pathogenesis of osteoarthritis. Yu et al. [35] showed that CAMP inhibited in ammation and promoted bone formation of bone marrow stromal cells (BMSC) through purinergic receptor P2 × 7(P2RX7) and mitogen-activated protein kinase (MAPK) signaling pathway. Further in vitro studies had found that CAMP gene methylation inhibited the level of ROS and TNF-α in chondrocytes, promoted the level of TGF-β expression, inhibited the proliferation of chondrocytes, and promoted their apoptosis. The results of Kuensaen et al. [36] found that high levels of CAMP promote the expression of downstream pro-in ammatory cytokines (especially IL17A), which was related to the pathogenesis of in ammatory arthritis. Hu et al. [37] found that CAMP regulated the production of in ammatory cytokines such as TNF-α and inhibited cell apoptosis. The results of this study suggested that the expression of CAMP gene and methylation in promoter region might be involved in the occurrence of osteoarthritis from the level of oxidative stress and the expression of in ammatory factors.
There are some limitations in this study. First of all, the treatment of the articular cartilage tissue samples used for analysis is unclear, and whether there are drugs that affect the expression of in ammatory factors in tissue is unclear. Secondly, the expression and methylation of CAMP gene may lead to the occurrence of OA through other signaling pathways, which was not con rmed in this study. In addition, the results of this study need to be further con rmed in vivo.

Conclusion
In this study, we found that CAMP gene promoter methylation induces chondrocyte apoptosis by inhibiting ROS levels and in ammatory response levels, which is a potential target for OA therapy.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
Not applicable Availability of data and materials The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
Competing interests Figure 1 Transcriptome and methylation level detection in OA patients. a, Analysis of the relative expression level of CAMP mRNA in the GSE117999 dataset, ncontrol=10, nOA=10. b, The methylation level of CAMP gene in the GSE48422 dataset, ncontrol=4, nOA=4.