Zhao Lei, Jiao Jiaxun, Yan Guanghui, Wei Wei, Fang Genqiang, Yu Tiemiao
Department of Spinal Surgery, Harrison International Peace Hospital, Hengshui City, Hebei Province, China.
Regen Ther. 2022 Jul 18;21:175-184. doi: 10.1016/j.reth.2022.06.005. eCollection 2022 Dec.
Circular RNAs (circRNAs) play a crucial regulatory role in human diseases. However, the roles of circRNAs in ankylosing spondylitis (AS) are barely known. In this study, the functions of circ_0018168 in AS were investigated.
Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assay were used for circ_0018168, microRNA-330-3p (miR-330-3p), dickkopf-1 (DKK1), alkaline phosphatase (ALP), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2) levels. Cell Counting Kit-8 (CCK-8) assay and 5'-ethynyl-2'-deoxyuridine (EdU) assay were conducted to analyze cell proliferation ability. Flow cytometry analysis was manipulated for cell cycle process. ALP activity was examined with a commercial kit. RNA immunoprecipitation (RIP) assay, RNA pull-down assay and dual-luciferase reporter assay were used to analyze the relationships of circ_0018168, miR-330-3p and DKK1.
Circ_0018168 and DKK1 levels were lowly expressed in AS hip capsule specimens. Circ_0018168 overexpression repressed cell proliferation, cell cycle process as well as reduced ALP activity and ALP, OCN and Runx2 protein levels in AS fibroblasts. DKK1 silencing ameliorated the impact of circ_0018168 on AS progression. In addition, circ_0018168 served as the sponge for miR-330-3p, which could target DKK1. MiR-330-3p inhibition suppressed the proliferation, cell cycle and osteogenic differentiation in AS fibroblasts, but DKK1 silencing reversed the impacts. Besides, the effect of circ_0018168 on AS development was abolished by miR-330-3p upregulation.
Circ_0018168 overexpression restrained fibroblast proliferation and osteogenic differentiation in AS by elevating DKK1 through adsorbing miR-330-3p.
环状RNA（circRNAs）在人类疾病中发挥着关键的调节作用。然而，circRNAs在强直性脊柱炎（AS）中的作用却鲜为人知。在本研究中，我们探究了circ_0018168在AS中的功能。
采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测circ_0018168、微小RNA-330-3p（miR-330-3p）、Dickkopf-1（DKK1）、碱性磷酸酶（ALP）、骨钙素（OCN）、 runt相关转录因子2（Runx2）的水平。使用细胞计数试剂盒-8（CCK-8）法和5'-乙炔基-2'-脱氧尿苷（EdU）法分析细胞增殖能力。通过流式细胞术分析细胞周期进程。使用商业试剂盒检测ALP活性。采用RNA免疫沉淀（RIP）法、RNA下拉法和双荧光素酶报告基因测定法分析circ_0018168、miR-330-3p和DKK1之间的关系。
circ_0018168和DKK1水平在AS髋关节囊标本中低表达。circ_0018168过表达抑制了AS成纤维细胞的增殖、细胞周期进程，并降低了ALP活性以及ALP、OCN和Runx2蛋白水平。DKK1沉默改善了circ_0018168对AS进展的影响。此外，circ_0018168作为miR-330-3p的海绵，miR-330-3p可靶向DKK1。抑制miR-330-3p可抑制AS成纤维细胞的增殖、细胞周期和骨分化，但DKK1沉默可逆转这些影响。此外，上调miR-330-3p可消除circ_0018168对AS发展的影响。
circ_0018168过表达通过吸附miR-330-3p上调DKK1，从而抑制AS中纤维母细胞的增殖和骨分化。
