Supplementary Material for: NCOA3 Loss Disrupts Molecular Signature of Chondrocytes and Promotes Posttraumatic Osteoarthritis Progression

<b><i>Background/Aims:</i></b> Osteoarthritis (OA) is the most common joint disease. Recently, a novel variant near the nuclear receptor coactivator 3 (<i>NCOA3</i>) has been identified in association with greater risk of developing OA. However, how NCOA3 is regulated in chondrocytes and involved in OA pathogenesis remain elusive. <b><i>Methods:</i></b> The expression and DNA methylation of <i>NCOA3</i> in knee OA cartilage and <i>in vitro</i> dedifferentiated chondrocytes with or without rs6094710 SNP were analyzed by qRT-PCR, immunoblotting, methylation-specific PCR and bisulfite sequencing. NCOA3 was depleted by siRNA or shRNA or inhibited by a chemical inhibitor to assess its role in chondrocyte dedifferentiation or OA pathogenesis in posttraumatic OA animal model established by cruciate ligament transection surgery. <b><i>Results:</i></b> We found that compared with normal counterparts, samples with rs6094710 SNP failed to upregulate NCOA3. Further evidence associated this phenotype with DNMT1-mediated hypermethylation in gene promoter region. Moreover, we showed that NCOA3 maintained the molecular signature of chondrocytes dedifferentiating <i>in vitro</i> or exposed to IL-1β, nevertheless, NCOA3 appeared dispensable for preventing OA initiation, since NCOA3 loss did not trigger OA in young mice. Instead, NCOA3 loss promoted posttraumatic OA progression, and in parallel, enhanced NF-κB activation. Finally, the promoted posttraumatic OA progression was significantly retarded when administrated with NF-κB pathway inhibitor, suggesting that NCOA3 lose promotes posttraumatic OA at least partially by enhancing NF-κB activation. <b><i>Conclusion:</i></b> Thus, our findings indicate a critical role of NCOA3 in chondrocytes, and imply that manipulating NCOA3 might present a potential therapeutic approach to interfere OA progression.

<b><i>背景与目的：</i></b> 骨关节炎（Osteoarthritis, OA）是最常见的关节疾病。近期研究发现，核受体辅激活因子3（nuclear receptor coactivator 3, NCOA3）基因附近的新型变异与OA发病风险升高显著相关。然而，软骨细胞中NCOA3的调控机制及其参与OA发病的具体过程仍不明确。<b><i>方法：</i></b> 本研究通过实时荧光定量聚合酶链反应（qRT-PCR）、免疫印迹（immunoblotting）、甲基化特异性PCR（methylation-specific PCR）及亚硫酸氢盐测序（bisulfite sequencing），分析了膝OA软骨组织以及携带或不携带rs6094710单核苷酸多态性（single nucleotide polymorphism, SNP）的<i>体外</i>去分化软骨细胞中NCOA3的表达与DNA甲基化水平。通过小干扰RNA（siRNA）或短发夹RNA（shRNA）敲低NCOA3，或使用化学抑制剂抑制其功能，以评估其在软骨细胞去分化中的作用，以及在交叉韧带切断术构建的创伤后OA动物模型中对OA发病的影响。<b><i>结果：</i></b> 研究发现，与正常对照样本相比，携带rs6094710 SNP的样本无法上调NCOA3的表达。进一步研究证实，该表型与基因启动子区域内DNA甲基转移酶1（DNMT1）介导的高甲基化密切相关。此外，本研究证明NCOA3可维持软骨细胞在<i>体外</i>去分化或经白细胞介素1β（IL-1β）处理后的分子特征；但NCOA3似乎并非OA起始的必需因子，因为年轻小鼠中NCOA3缺失并不会诱发OA。相反，NCOA3缺失会促进创伤后OA的进展，并同步增强核因子κB（NF-κB）通路的激活。最终，给予NF-κB通路抑制剂可显著延缓创伤后OA的进展，提示NCOA3缺失至少部分通过增强NF-κB通路激活来促进创伤后OA的发生发展。<b><i>结论：</i></b> 综上，本研究结果揭示了NCOA3在软骨细胞中的关键作用，并提示调控NCOA3的表达或功能可能成为干预OA进展的潜在治疗策略。