METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

Abstract METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.


| INTRODUC TI ON
At present, spinal degenerative disease is one of the most common causes of a declining labour force and quality of life globally. Thus, it brings a very heavy economic burden not only to families, but also to society. [1][2][3] Disc degeneration is the pathological basis of the occurrence and development of spinal degenerative diseases. Therefore, it is particularly important to understand the pathophysiological process of disc degeneration and actively take effective intervention measures to prevent and treat disc degeneration and related diseases. 4 Endplate cartilage, a thin layer of transparent cartilage between the intervertebral disc and adjacent vertebral body, plays important roles in nutrient exchange in the nucleus pulposus, stress buffering of the intervertebral disc and maintenance of the normal shape of the adjacent vertebral body. [5][6][7] Previous studies have shown that endplate cartilage degeneration is one of the causes for disc degeneration, and maintenance of the normal function of endplate cartilage plays a decisive role in the prevention and treatment of disc degeneration. 8 Inflammatory responses are one of the most important inducing factors for the degeneration of endplate chondrocytes. First, they affect the quality of endplate cartilage matrix by regulating matrix synthesis efficiency and the activity of proteases. In addition, they regulate the growth, differentiation and apoptosis of chondrocytes through the cytokine network. 9 At present, few studies have investigated the relationship between methylation of m6A and cartilage degeneration. 15

| Safranin O staining
Sixty nine endplate cartilage specimens were fixed, embedded and sectioned in the sagittal plane with a thickness of 5 μm. After gradient dewaxing, paraffin sections were incubated with Safranin O staining solution (Gibco, Thermo Fisher Scientific), and histological changes were observed under a light microscope after clearing.

| Detection of cell proliferation and apoptosis
To assay cell proliferation, treated chondrocytes were seeded into 96-well plates at a density of 5 × 10 3 cells per well. Freshly prepared Cell Counting Kit-8 solution (Dojindo Molecular Technologies, Tokyo, Japan) 10 μL was added to the plates containing cells for incubation at 37°C for 2 hours. After seed, the plates were vortexed for 2 minutes and the absorbance of each well was measured at 450 nm using a spectral scanning multimode microplate reader (Bio-Rad).

| Gene transfection
Target-specific siRNAs were cloned into a lentivirus vector and transfected into endplate chondrocytes. Transfected cells were screened by puromycin (4 μg/mL) and then amplified to form stable sublines, which were used as a negative control (siCtrl).

| Reverse transcription quantitative polymerase chain reaction (RT-QPCR)
According to the manufacturer's instructions, total RNA in endplate chondrocytes was extracted with TRIzol (Invitrogen).
miRNA and mRNA expression levels were detected with a

| Western blot
Proteins in endplate chondrocytes were lysed and collected, and the protein concentration was determined by the bicinchoninic acid quantitative method. Denatured protein was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis at 20 g per pore and then transferred onto a nitrocellulose membrane.
The membrane was blocked with 5% bovine serum albumin for were added. The membrane was then incubated at 4°C overnight.
The following day, a secondary antibody (1:5000; Cell Signaling Technology, Danvers, MA) was added and the membrane was incubated again for 1 hour at room temperature on a shaker. After washing the membrane three times with Tris-buffered saline containing Tween 20, images were acquired with a gel imaging system.

| Immunohistochemical staining
Paraffin sections of cartilage tissue were prepared, embedded, trimmed and sliced with a vibratome into 5-μm sections. After drying, dewaxing and gradient alcohol hydration, antigen repair was carried out. Anti-IL-1β (1:100, Abcam) was added and incubated overnight in a 37°C incubator. After washing with Tris-buffered saline, the secondary antibody was added, and DAB staining and haematoxylin re-staining were performed.

| CO-immunoprecipitation (CO-IP)
According to the instructions of a Pierce TM Co-IP Kit (Thermo  Figure 1A). RT-qPCR, Western blot and colorimetry showed that with the gradual deterioration of endplate cartilage, expression of the m6A RNA methyltransferase METTL3 was increased, the overall methylated level of m6A was increased, and the two showed a low degree of positive correlation ( Figure 1B-F).
These results indicate that METTL3-mediated methylation of m6A occurred in human endplate cartilage.

| IL-1β can induce degeneration of endplate chondrocytes
Immunohistochemistry was used to detect IL-1β in human endplate cartilage with different degrees of degeneration. Using image J software to analyse the percentage of IL-1β protein staining part in the whole image area, we found that the higher the degree of degeneration, the higher the expression level of IL-1β in cartilage tissue (Figure 2A), suggesting that IL-1β may play an important role in the degeneration of endplate cartilage. Next, we digested the human endplate cartilage and extracted primary cells for culture ( Figure 2B). In our previous study, we observed the effects of different concentrations of IL-β (0 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/ mL and 20 ng/mL) on chondrocytes. The results showed that IL-β more than 10 ng/mL was prone to a large number of cell death.
We speculate that this may be due to the low viability of cells derived from human degenerative endplate. Therefore, in this study, we only observed the effects of IL-β on chondrocytes at 0 ng/mL,

| MIR-126 inhibited the PI3K/AKT pathway by down-regulating PIK3R2 expression in response to IL-1β
Previous studies have shown that METTL3 regulates miR-126 maturation, so we examined the role of miR-126-5p in IL-1β-induced degeneration of endplate chondrocytes and its molecular mechanism. First, we found that IL-1β induced up-regulation of miR-126-5p expression ( Figure 4A). Next, we examined the effect of
On contrary, after METTL3 overexpression, miR-126-5p expression was significantly increased and pri-miR-126-5p expression was significantly decreased ( Figure 5C-G). Using immunoprecipitation, we found that METTL3 can bind to DGCR8 protein ( Figure 5H). In addition, DGCR8 inhibition was consistent with the result of METTL3 inhibition, miR-126-5p expression was significantly decreased and pri-miR-126-5p expression was significantly increased ( Figure 5I-M). Besides, we examined human endplate cartilage and found that the expression level of METTL3 was positively correlated with miR-126-5p ( Figure 5N). These results indicated that METTL3 was involved in pri-miR-126-5p processing and promoted miR-126-5p maturity.

F I G U R E 5
The effect of methylated modification mediated by METTL3 on the maturation of mir-126-5p. A and B, RT-qPCR was used to detect change of miR-126 and pri-miR-126 expression after METTL3 inhibition. C and D, RT-qPCR was used to detect change of miR-126 and pri-miR-126 expression after METTL3 overexpression. E, F and G, The overexpression efficiency of METTL3 was detected by RT-qPCR and Western blot. H, Co-IP was used to detect the combination between METTL3 and DGCR8. I, J and K, The knockdown efficiency of DGCR8 was detected by RT-qPCR and Western blot. L and M, RT-qPCR was used to detect change of miR-126 and pri-miR-126 expression after DGCR8 inhibition. N, Pearson test was used to analyse the correlation between the expression level of METTL3 and miR-126. O, Diagram of the mechanism. (**P < .01)

| CON CLUS ION
In brief, this study revealed the interaction among METTL3, miR-126-5p and PIK3R2 in endplate chondrocytes under the action of IL-1β ( Figure 5O). Specifically, we found that IL-1β up-regulated METTL3 expression in endplate chondrocytes. Also, METTL3 promoted the up-regulation of miR-126-5p expression by m6A methylation modification, and subsequently suppressed the protective effect of the PI3K/Akt pathway on endplate chondrocytes through its target gene PIK3R2, resulting in dysfunctional cell vitality and metabolism, and ultimately the degeneration of endplate chondrocytes.

CO N FLI C T O F I NTE R E S T
No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication. All the authors listed have approved the manuscript that is enclosed.

E TH I C A L A PPROVA L
All experimental procedures, specimen acquisition in this study was reviewed and approved by the ethics committee of Yijishan Hospital of Wannan Medical College (Wuhu, China). All surgical patients participating in the study were aware of the study and signed the informed consent forms.

DATA AVA I L A B I L I T Y S TAT E M E N T
According to the requirements, data can be obtained from the corresponding authors to support the results of this study.