Abstract

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.

摘要

本研究的目的是探讨 hsa_circ_0056558/miR-1290/CDK6 轴对强直性脊柱炎 (AS) 的影响。分别基于RNA-seq数据和微阵列数据分析AS组织中差异表达的has_circ_0056558和miR-1290。进行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。进行蛋白质印迹分析以测试 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质水平。进行CCK8测定和流式细胞术分别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。通过碱性磷酸酶 (ALP) 活性测定分析成纤维细胞的分化。我们的研究结果表明 circ_0056558 和 CDK6 在 AS 组织中的表达水平均显着高于正常样本。此外，hsa_circ_0056558 可以通过促进 CDK6 表达和抑制 miR-1290 在 AS 中的表达来抑制细胞增殖和分化。miR-1290 的过表达负调节 CDK6 表达以增强细胞增殖。p-AKT、p-NF-κB p65 和 p-IκBα 的蛋白质水平受到 hsa_circ_0056558 或 CDK6 过表达的促进，同时受到 miR-1290 上调的抑制。综上所述，