破骨细胞内microRNA-214-3p介导骨关节炎早期软骨下骨骨重塑失常的分子机制研究

Emerging evidence suggests that the aberrant osteoclast-mediated subchondral bone (SCB) remodeling precedes the articular cartilage degradation and plays a critical role in early-stage osteoarthritis (OA). However, the effect of anti-resorptive agents on clinical treatment for OA cartilage degradation is still controversial, indicating the requirement of further investigation on molecular mechanism responsible for the aberrant subchondral bone remodeling in early-stage OA. We have previously established the functional role of microRNA-214-3p (miR-214-3p) in bone remodeling. Recently, we further observed that the expression level of miR-214-3p within the SCB osteoclasts was upregulated in early-stage OA, which preceded the increase in the serum bone resorption marker and cartilage degradation marker. Moreover, our in vitro data showed that osteoclastic miR-214-3p could directly target TIMP2 (TIMP metallopeptidase inhibitor 2, a tissue inhibitor of metalloproteinases) and promote the degradation of co-cultured cartilage explant. Thus, we hypothesized that the elevated osteoclastic miR-214-3p could mediate the aberrant SCB remodeling to contribute to early OA development. To facilitate testing the hypothesis, we have generated the osteoclast-specific miR-214-3p conditional knockout / knockin mice, and developed an osteoclast-targeted delivery system for evaluating the role of osteoclastic miR-214-3p in early OA development by genetic and pharmacological approaches, respectively. The proposed study would help to uncover the underlying molecular mechanism responsible for the aberrant SCB remodeling and cartilage degradation in early-stage OA. It might also indicate novel therapeutic targets for early treatment of OA.

以破骨细胞介导的骨吸收亢进为主要特征的软骨下骨骨重塑失常是骨关节炎早期的重要病理改变，其分子机制尚不明确。我们此前证实微小核糖核酸miR-214-3p可调控骨重塑。我们近期在骨关节炎小鼠模型中进一步发现软骨下破骨细胞内的miR-214-3p显著升高，且早于骨吸收和软骨退变标志物的升高。体外实验显示破骨细胞miR-214-3p可抑制TIMP2并促进共培养软骨块的降解。因此我们假设破骨细胞内高表达的miR-214-3p介导了骨关节炎早期软骨下骨骨重塑失常。我们拟用切除膝关节前交叉韧带的骨关节炎小鼠模型来验证上述假设，利用已建立的破骨细胞特异性miR-214-3p敲除／敲入的基因工程小鼠以及破骨细胞靶向递送系统等工具来研究干预破骨细胞内miR-214-3p对软骨下骨骨重塑及软骨退变的影响。本研究可望阐释骨关节炎早期软骨下骨骨重塑失常的分子机制，为早期干预骨关节炎提供新的治疗策略。

以破骨细胞介导的骨吸收亢进为主要特征的软骨下骨骨重塑失常是骨关节炎早期的重要病理改变，其分子机制尚不明确。我们的前期研究发现微小核酸miR-214-3p可调控骨重塑，且在骨关节炎小鼠模型的软骨下骨破骨细胞内的miR-214-3p显著升高，且早于骨吸收和软骨退变标志物的升高。体外实验显示破骨细胞miR-214-3p可抑制TIMP2并促进共培养软骨块的降解。另外，我们的体外研究还发现破骨细胞分泌的外泌体包裹包括miR-214-3p在内的一系列微小核酸可介导破骨细胞-软骨细胞通讯并抑制软骨细胞内TIMP2的表达。因此，我们假设破骨细胞内高表达的微小核酸（miR-214-3p)介导了骨关节炎早期骨重塑和软骨退变。本研究中，我们构建切除前交叉韧带的骨关节炎小鼠模型，分别运用破骨细胞特异性miR-214-3p敲除/敲入的基因工程小鼠验证我们的假设。研究结果显示:（1）miR-214-3p CKO小鼠的OA软骨退变程度以及软骨下骨与软骨交界处的血管和交感神经轴突侵入程度较野生型小鼠显著缓解，而miR-214-3p CKI小鼠的的OA软骨退变程度以及软骨下骨与软骨交界处的血管和交感神经轴突侵入程度较野生型小鼠显著恶化，而靶向静默破骨细胞内Rab27a蛋白阻断其外泌体释放可显著改善CKI小鼠的OA病变。（2）全身注射正常表达miRNA的破骨细胞外泌体相比miRNA敲低的破骨细胞外泌体可显著促进OA软骨退变程度以及软骨下骨与软骨交界处的血管和交感神经轴突侵入程度。而过表达关节软骨的TIMP2后，可显著缓解因注射正常表达miRNA的破骨细胞外泌体而恶化的OA软骨病变。上述研究发现证实破骨细胞释放包含miR-214-3p的外泌体通过调控软骨细胞TIMP2参与贡献了OA软骨退变。本研究揭示了一种由外泌体微小核酸介导的破骨-软骨细胞通讯新机制，可望为OA的新药研发提供新的思路与策略。