Targeting long non-coding RNA H19 in Subchondral Bone Osteocytes Alleviates Cartilage Degradation in Osteoarthritis

Introduction: Emerging evidence suggests long non-coding RNA (lncRNA) H19 is associated with osteoarthritis (OA) pathology. However, how H19 contributes to OA has not been reported. This study aims to investigate the biological function of H19 in OA subchondral bone remodeling and OA progression. Methods: Clinical joint samples and OA animal models induced by medial meniscus destabilization (DMM) surgery were used to verify the causal relationship between osteocyte H19 and OA subchondral bone and cartilage changes. MLO-Y4 osteocyte cells subjected to fluid shear stress were used to verify the mechanism underlying H19-mediated mechano-response. Finally, the antisense oligonucleotide (ASO) against H19 was delivered to mice knee joints by magnetic metal-organic framework (MMOF) nanoparticles in order to develop a site-specific delivery method for targeting osteocyte H19 for OA treatment. Results: Both clinical OA subchondral bone and wildtype mice with DMM-induced OA exhibit aberrant higher subchondral bone mass with more H19 expressing osteocytes. On the contrary, osteocyte-specific deletion of H19 mice is less vulnerable to DMM-induced OA phenotype. In MLO-Y4 cells, H19-mediated osteocyte mechano-response through PI3K/AKT/GSK3 signals activation by EZH2-induced H3K27me3 regulation on PP2A inhibition. Targeted inhibition of H19 (using ASO-loaded MMOF) substantially alleviates subchondral bone remodeling and OA phenotype. Discussion: In summary, our results provide new evidence that the elevated H19 expression in osteocytes may contribute to aberrant subchondral bone remodeling and OA progression. H19 appears to be required for the osteocyte response to mechanical stimulation, and targeting H19 represents a new promising approach for OA treatment. Overall design: To investigate the role of H19 in the regulation of osteocyte mechanotransduction, we establish MLO-Y4 cell lines in which target gene could be knocked down by ASO and throughout FSS We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 different cells Comparative gene expression profiling analysis of RNA-seq data for ASO-treated and control MLO-Y4 cells

引言：越来越多的证据表明，长链非编码RNA（long non-coding RNA，lncRNA）H19与骨关节炎（osteoarthritis，OA）的病理进程相关。然而，目前尚未有研究阐明H19参与骨关节炎发病的具体机制。本研究旨在探究H19在骨关节炎软骨下骨重塑及疾病进展中的生物学功能。

方法：本研究采用临床关节样本以及经内侧半月板失稳术（medial meniscus destabilization，DMM）构建的骨关节炎动物模型，验证骨细胞中H19与骨关节炎软骨下骨及软骨病变之间的因果关联。本研究使用经流体剪切应力处理的MLO-Y4骨细胞系，验证H19介导机械应答的潜在分子机制。最后，本研究通过磁性金属有机框架（magnetic metal-organic framework，MMOF）纳米颗粒将靶向H19的反义寡核苷酸（antisense oligonucleotide，ASO）递送至小鼠膝关节，以开发针对骨细胞H19的骨关节炎靶向递送治疗策略。

结果：临床骨关节炎患者的软骨下骨组织以及经DMM诱导骨关节炎的野生型小鼠，均表现出异常升高的软骨下骨骨量，且表达H19的骨细胞数量增多。与之相反，特异性敲除骨细胞中H19的小鼠，对DMM诱导的骨关节炎表型的易感性显著降低。在MLO-Y4细胞中，H19通过EZH2介导的组蛋白H3第27位三甲基化（H3K27me3）调控蛋白磷酸酶2A（PP2A）的抑制作用，进而激活PI3K/AKT/GSK3信号通路，介导骨细胞的机械应答反应。采用装载ASO的MMOF靶向抑制H19，可显著改善软骨下骨重塑及骨关节炎表型。

讨论：综上，本研究结果提供了新的证据：骨细胞中H19表达上调可能参与异常软骨下骨重塑及骨关节炎的疾病进展。H19是骨细胞响应机械刺激所必需的分子，靶向H19有望成为骨关节炎治疗的全新策略。

总体实验设计：为探究H19在骨细胞机械转导调控中的作用，本研究构建了可通过ASO敲低靶基因的MLO-Y4细胞系，并施加流体剪切应力（fluid shear stress，FSS）处理。随后，基于两种不同细胞的RNA测序数据进行基因表达谱分析；同时对ASO处理组与对照组MLO-Y4细胞的RNA测序数据开展对比基因表达谱分析。