Rheumatology and Immunology Department, The Second People's Hospital of Liaocheng, The Second Hospital of Liaocheng Affiliated to Shandong First Medical University, Linqing, P.R. China.
Liaocheng Dongchangfu District Maternal and Child Health Hospital, Liaocheng, P.R. China.
Autoimmunity. 2021 Mar;54(2):114-128. doi: 10.1080/08916934.2021.1894417. Epub 2021 Mar 9.
The aims of this study was to investigate the influences of hsa_circ_0056558/miR-1290/CDK6 axis in ankylosing spondylitis (AS). The differentially expressed has_circ_0056558 and miR-1290 in AS tissue were analysed based on RNA-seq data and microarray data, respectively. qRT-PCR was performed for detection of relative expression levels of hsa_circ_0056558, miR-1290, CDK6, osteogenic differentiation markers (Runx2 and Osterix) and other inflammatory factors (TNF-α, IL-1β, and IL-6). Western blotting analysis was conducted to test the protein levels of CDK6, osteogenic differentiation markers (Runx2 and Osterix), and PI3K/AKT/NF-κB pathway-associated proteins. CCK8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability, respectively. Targeted relationships were predicted by bioinformatic analysis and verified by dual-luciferase reporter assay. The differentiation of fibroblast cells was analysed by alkaline phosphatase (ALP) activity assay. Our findings revealed that the expression levels of both circ_0056558 and CDK6 in AS tissue were significantly higher than that in normal samples. Besides, hsa_circ_0056558 could suppress cell proliferation and differentiation by facilitating CDK6 expression and suppressing miR-1290 expression in AS. Over-expression of miR-1290 negatively regulated CDK6 expression to enhance cell proliferation. The protein levels of p-AKT, p-NF-κB p65, and p-IκBα were promoted by hsa_circ_0056558 or CDK6 over-expression while suppressed by miR-1290 up-regulation. In conclusion, our study demonstrated that hsa_circ_0056558 and CDK6 suppressed cell proliferation and differentiation while enhanced cell apoptosis by competitive binding to miR-1290 in AS, which might be possibly achieved by PI3K/AKT/NF-κB pathway, providing us novel therapeutic strategy for AS.
本研究旨在探讨 hsa_circ_0056558/miR-1290/CDK6 轴在强直性脊柱炎（AS）中的影响。基于 RNA-seq 数据和微阵列数据，分别分析了 AS 组织中差异表达的 hsa_circ_0056558 和 miR-1290。通过 qRT-PCR 检测 hsa_circ_0056558、miR-1290、CDK6、成骨分化标志物（Runx2 和 Osterix）和其他炎症因子（TNF-α、IL-1β 和 IL-6）的相对表达水平。Western blot 分析检测 CDK6、成骨分化标志物（Runx2 和 Osterix）和 PI3K/AKT/NF-κB 通路相关蛋白的蛋白水平。CCK8 测定和流式细胞术分别用于测定细胞增殖和细胞凋亡能力。通过生物信息学分析预测靶向关系，并通过双荧光素酶报告基因检测进行验证。碱性磷酸酶（ALP）活性测定分析成纤维细胞分化。我们的研究结果表明，AS 组织中 circ_0056558 和 CDK6 的表达水平明显高于正常样本。此外，hsa_circ_0056558 可通过促进 CDK6 表达和抑制 miR-1290 表达来抑制 AS 中的细胞增殖和分化。miR-1290 的过表达负调控 CDK6 表达，增强细胞增殖。hsa_circ_0056558 或 CDK6 的过表达促进 p-AKT、p-NF-κB p65 和 p-IκBα 的蛋白水平，而 miR-1290 的上调抑制 p-AKT、p-NF-κB p65 和 p-IκBα 的蛋白水平。综上所述，本研究表明，hsa_circ_0056558 和 CDK6 通过与 miR-1290 竞争结合来抑制 AS 中的细胞增殖和分化，同时增强细胞凋亡，这可能通过 PI3K/AKT/NF-κB 通路实现，为 AS 提供了新的治疗策略。
