LncRNA MIR22HG promotes osteoarthritis progression via regulating miR-9-3p/ADAMTS5 pathway

Dysregulation of long non-coding RNAs (lncRNAs) plays a fundamental role in the development and progression of osteoarthritis (OA), but the potential functions of lncRNAs in OA were not fully clarified. In the present work, we want to clarify the underlying functions and mechanisms of MIR22HG in OA. qRT-PCR was employed to detect the mRNA expression of MIR22HG, miR-9-3p, and ADAMTS5, while the protein expressions were measured using Western blot. The cell proliferation was examined through CCK8, while apoptosis was used in flow cytometry. Luciferase reporter assay and RNA immunoprecipitation (RIP) assays were undertaken to investigate the binding relationship among MIR22HG, ADAMTS5, and miR-9-3p. MIR22HG was significantly overexpressed in OA cartilages, OA chondrocytes and IL-1β-induced chondrocytes. Functionally, MIR22HG knockdown promoted cell proliferation, suppressed apoptosis, and contributed to downregulation of MMP13 and ADAMTS5 and upregulation of COL2A1 and ACAN in IL-1β-stimulated chondrocytes. Mechanistically, bioinformatic analysis indicated that MIR22HG may serve as a sponge for miR-9-3p and ADAMTS5 may be a potential targeted gene for miR-9-3p, which were subsequently verified through a dual-luciferase reporter assay. Moreover, rescue experiments showed that MIR22HG participated in the regulation of chondrocytes proliferation, apoptosis, and degradation of extracellular matrix via miR-9-3p/ADAMTS5 pathway. In conclusion, our findings illuminated that inhibition of MIR22HG ameliorated IL-1β-induced apoptosis and ECM degradation of human chondrocytes through miR-9-3p/ADAMTS5 pathway, which may provide a potentially promising target for OA treatment.

长链非编码RNA（long non-coding RNAs，lncRNAs）的表达失调在骨关节炎（osteoarthritis，OA）的发生与进展中发挥关键性作用，但目前lncRNAs在OA中的潜在功能尚未完全阐明。本研究旨在明确MIR22HG在OA中的潜在功能与作用机制。本研究采用实时定量聚合酶链反应（qRT-PCR）检测MIR22HG、miR-9-3p及ADAMTS5的mRNA表达水平，采用蛋白质印迹法（Western blot）检测相关蛋白表达水平；通过CCK-8法检测细胞增殖能力，流式细胞术（flow cytometry）检测细胞凋亡情况；利用荧光素酶报告基因实验（Luciferase reporter assay）与RNA免疫沉淀实验（RNA immunoprecipitation，RIP）探究MIR22HG、ADAMTS5与miR-9-3p之间的结合关系。研究结果显示，MIR22HG在OA软骨组织、OA软骨细胞及白细胞介素-1β（IL-1β）诱导的软骨细胞中均显著高表达。功能实验表明，在IL-1β刺激的软骨细胞中，敲低MIR22HG可促进细胞增殖、抑制细胞凋亡，并下调基质金属蛋白酶13（MMP13）与ADAMTS5的表达，同时上调Ⅱ型胶原α1链（COL2A1）与聚集蛋白聚糖（ACAN）的表达。机制层面，生物信息学分析（bioinformatic analysis）提示MIR22HG可作为miR-9-3p的分子海绵，且ADAMTS5可能是miR-9-3p的潜在靶基因，上述结论随后通过双荧光素酶报告基因实验（dual-luciferase reporter assay）得以验证。此外，挽救实验（rescue experiments）证实，MIR22HG可通过miR-9-3p/ADAMTS5通路调控软骨细胞的增殖、凋亡及细胞外基质（extracellular matrix，ECM）降解。综上，本研究结果表明，抑制MIR22HG可通过miR-9-3p/ADAMTS5通路改善IL-1β诱导的人软骨细胞凋亡与细胞外基质降解，该发现可为OA的治疗提供潜在的新型靶点。