Department of Rheumatology and Immunology, The Second Hospital of Tianjin Medical University, Tianjin, China.
Department of Knee Joint, Tianjin Hospital, Tianjin, China.
Immunol Invest. 2022 Aug;51(6):1529-1547. doi: 10.1080/08820139.2021.1981930. Epub 2021 Sep 21.
Circular RNAs (circRNAs) titrate the function of microRNAs (miRNAs), regulate transcription, and interfere with splicing. This study attempted to confirm the role of a novel circRNA circ_0128846 during osteoarthritis (OA) progression. Tissues and chondrocytes were isolated from OA patients. Overexpression and knockdown of target genes were generated using cell transfection and siRNA interference. Expression levels of genes were measured by qRT-PCR, Western blot, and immunohistochemistry, respectively. The interactions among circ_0128846, miR-140-3p, and JAK2 were verified by bioinformatics prediction, a dual-luciferase reporter assay, and RNA immunoprecipitation assay. The role of circ_0128846 in vivo was confirmed by the construction of experimental OA rats. Pathological changes were evaluated by hematoxylin and eosin and Safranin O staining. In OA patients, the level of circ_0128846 and JAK2 were up-regulated with down-regulated level of miR-140-3p. Circ_0128846 was principally located in the cytoplasm. Circ_0128846 silence enhanced cells viability, but reduced apoptosis rate and inflammatory response, which was obviously reversed by miR-140-3p knockdown. The overexpression of JAK2 reversed the effects of miR-140-3p on cell phenotypes. Circ_0128846 silence suppressed the level of MMP-13 and promoted the expression of collagen II by up-regulating miR-140-3p and down-regulating JAK2 in OA cells. Results of animal experiments demonstrated that circ_0128846 silence promoted collagen II expression and attenuated the OA progression by regulating the miR-140-3p/JAK2 axis. Circ_0128846 contributes to OA development through acting as a sponge RNA for miR-140-3p and thereby increasing JAK2 expression. Results indicated that targeting circ_0128846 may have the potential to alleviate OA progression.circRNAs: Circular RNAs; miRNAs: microRNAs; OA: osteoarthritis; RIP: RNA immunoprecipitation; H&E: hematoxylin and eosin; ncRNAs: noncoding RNAs; ceRNA: competitive endogenous RNA; DMEM: Dulbecco's modified Eagle's medium; PBS: phosphate buffered saline; OE-circ_0128846: overexpression vector for circ_0128846; pcDNA3.1-JAK2: pcDNA3.1 overexpression vector for Janus kinase 2; NC: negative control; CCK-8: Cell Counting Kit-8; PI: propidium iodide; WT: Wild-type; mutants (MUT); SD rats: Sprague Dawley rats; DMM: destabilization of medial meniscus; IHC: immunohistochemistry; DAB: diaminobenzene; pre-Mrna: precursor mRNA.
环状 RNA（circRNAs）调节 microRNA（miRNAs）的功能、调控转录并干扰剪接。本研究试图确认环状 RNA circ_0128846 在骨关节炎（OA）进展过程中的作用。从 OA 患者中分离组织和软骨细胞。通过细胞转染和 siRNA 干扰生成靶基因的过表达和敲低。通过 qRT-PCR、Western blot 和免疫组织化学分别测量基因的表达水平。通过生物信息学预测、双荧光素酶报告基因测定和 RNA 免疫沉淀测定验证 circ_0128846、miR-140-3p 和 JAK2 之间的相互作用。通过构建实验性 OA 大鼠来验证 circ_0128846 在体内的作用。通过苏木精和伊红以及番红 O 染色评估病理变化。在 OA 患者中，circ_0128846 和 JAK2 的水平上调，miR-140-3p 的水平下调。Circ_0128846 主要位于细胞质中。Circ_0128846 沉默增强了细胞活力，但降低了凋亡率和炎症反应，这一反应被 miR-140-3p 的敲低明显逆转。JAK2 的过表达逆转了 miR-140-3p 对细胞表型的影响。Circ_0128846 沉默通过上调 miR-140-3p 和下调 JAK2 抑制 OA 细胞中 MMP-13 的水平并促进胶原 II 的表达。动物实验结果表明，Circ_0128846 沉默通过调节 miR-140-3p/JAK2 轴促进胶原 II 表达并减轻 OA 进展。Circ_0128846 通过作为 miR-140-3p 的海绵 RNA 发挥作用，从而增加 JAK2 的表达，促进 OA 发生发展。结果表明，针对 circ_0128846 可能具有减轻 OA 进展的潜力。circRNAs：环状 RNA；miRNAs：microRNAs；OA：骨关节炎；RIP：RNA 免疫沉淀；H&E：苏木精和伊红；ncRNAs：非编码 RNA；ceRNA：竞争性内源性 RNA；DMEM：杜尔贝科改良 Eagle 培养基；PBS：磷酸盐缓冲盐水；OE-circ_0128846：circ_0128846 的过表达载体；pcDNA3.1-JAK2：Janus 激酶 2 的 pcDNA3.1 过表达载体；NC：阴性对照；CCK-8：细胞计数试剂盒-8；PI：碘化丙啶；WT：野生型；突变体（MUT）；SD 大鼠：Sprague Dawley 大鼠；DMM：内侧半月板不稳定；IHC：免疫组织化学；DAB：二氨基联苯胺；pre-Mrna：前体 mRNA。
