Objective

Tension stimulation is an important inducer of endplate cartilage degeneration, but the specific regulatory mechanism remains unclear. This study was the first to reveal the mechanism by which methyltransferase-like 3 (METTL3)-mediated N(6)-methyladenosine (m6A) modification affected the extracellular matrix anabolism by tension-induced endplate chondrocytes.

Method

We examined the differences in METTL3 expression and m6A methylation levels in human endplate chondrocytes and human cartilage endplate tissues under in vitro tension. The effect on endplate cartilage degeneration was evaluated by manipulating m6A methylation mediated by METTL3 in vivo and in vitro. The effect of METTL3-mediated m6A methylation on the stability of sex-determining region Y-box transcription factor 9 (SOX9) gene expression was determined experimentally.

Results

METTL3 expression and m6A methylation levels were significantly increased in degenerative human endplate cartilage tissue. Similarly, tension stimulation inhibited the ability of human endplate chondrocytes to synthesize extracellular matrix, which was accompanied by an increase in METTL3-mediated m6A methylation. The ability of endplate chondrocytes to resist tension was significantly enhanced by inhibiting METTL3 expression and subsequently downregulating m6A methylation in vitro and in vivo, thereby reducing intervertebral disc degeneration. Furthermore, METTL3 mediated SOX9 RNA methylation and disrupted SOX9 mRNA stability, thereby inhibiting the gene expression of the downstream collagen type II alpha 1 chain.

Conclusion

Tension stimulation downregulated SOX9 expression through METTL3-mediated m6A methylation, thereby inhibiting the synthesis of extracellular matrix in endplate chondrocytes.

中文翻译：

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N(6)-甲基腺苷 RNA 甲基转移酶样 3 通过在张力下下调性别决定区 Y-Box 转录因子 9 的表达来抑制终板软骨细胞的细胞外基质合成

客观的

张力刺激是终板软骨退变的重要诱因，但具体调控机制尚不清楚。这项研究首次揭示了甲基转移酶样 3 (METTL3) 介导的 N(6)-甲基腺苷 (m6A) 修饰影响张力诱导的终板软骨细胞细胞外基质合成代谢的机制。

方法

我们检查了体外张力下人终板软骨细胞和人软骨终板组织中 METTL3 表达和 m6A 甲基化水平的差异。通过在体内和体外操纵由 METTL3 介导的 m6A 甲基化来评估对终板软骨退化的影响。实验确定了 METTL3 介导的 m6A 甲基化对性别决定区 Y-box 转录因子 9 ( SOX9 ) 基因表达稳定性的影响。

结果

METTL3 表达和 m6A 甲基化水平在退行性人终板软骨组织中显着增加。同样，张力刺激抑制人终板软骨细胞合成细胞外基质的能力，同时伴随着 METTL3 介导的 m6A 甲基化增加。通过抑制 METTL3 表达并随后在体外和体内下调 m6A 甲基化，终板软骨细胞抵抗张力的能力显着增强，从而减少椎间盘退变。此外，METTL3 介导 SOX9 RNA 甲基化并破坏 SOX9 mRNA 稳定性，从而抑制下游 II 型胶原蛋白 α1 链的基因表达。

结论

张力刺激通过 METTL3 介导的 m6A 甲基化下调SOX9的表达，从而抑制终板软骨细胞中细胞外基质的合成。