环状RNA circ_0099630/微小RNA-940/受体相关因子6调控级联反应调节牙周炎的发病机制。
The circular RNA circ_0099630/miR-940/receptor-associated factor 6 regulation cascade modulates the pathogenesis of periodontitis.
作者信息
Zhao Xue-Qin, Ao Chuan-Bei, Yan Yi-Tong
机构信息
Department of Stomatology, Stomatological Hospital of Jingmen Second People's Hospital, Jingmen, China.
出版信息
J Dent Sci. 2022 Oct;17(4):1566-1576. doi: 10.1016/j.jds.2022.04.005. Epub 2022 Apr 25.
BACKGROUND/PURPOSE: Periodontitis is one of the highly prevalent chronic inflammatory conditions in adults. The importance of circular RNAs (circRNAs) in the regulation of inflammation has been gradually reported in recent years, but the role of circRNA circ_0099630 in periodontitis has not been reported.
MATERIALS AND METHODS
The contents of circ_0099630, microRNA-940 (miR-940) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Inflammatory factor secretion, cell proliferation, and apoptosis were analyzed under the application of Enzyme-linked immunosorbent assay (ELISA), Cell Counting Kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU) and flow cytometry, respectively. The Western blot also analyzed the phosphorylation levels of RELA proto-oncogene (P65) and IkappaBalpha (IκBα), key molecules of the nuclear factor kappa-B (NF-κB) pathway. The relationship between miR-940 and circ_0099630 or TRAF6 was verified by luciferase reporter system and RNA immunoprecipitation (RIP) assay.
RESULTS
Higher abundance of circ_0099630 and TRAF6 and lower miR-940 expression were observed in periodontitis, and circ_0099630 knockdown attenuated the damage of human PDL cells (PDLCs) induced by lipopolysaccharides (LPS). The relationship between miR-940 and circ_0099630 or TRAF6 was evidenced, while miR-940 downregulation diminished the repair effect of si-circ_0099630 on overexpression LPS-induced damage in PDLCs. Similarly, TRAF6 upregulation impaired the mitigating effect of miR-940 overexpression on LPS-induced injury in PDLCs. Circ_0099630 silencing evidently curbed the phosphorylation levels of P65 and IκBα and thus attenuating the inflammatory response by acting on the miR-940/TRAF6 axis.
CONCLUSION
Silencing circ_0099630 alleviates LPS-induced periodontal ligament cell injury via targeting miR-940/TRAF6/NF-κB in periodontitis.
背景/目的：牙周炎是成年人中高度流行的慢性炎症性疾病之一。近年来，环状RNA（circRNA）在炎症调节中的重要性已逐渐被报道，但circRNA circ_0099630在牙周炎中的作用尚未见报道。
材料与方法
采用定量实时聚合酶链反应（qRT-PCR）或蛋白质免疫印迹法检测circ_0099630、微小RNA-940（miR-940）和肿瘤坏死因子（TNF）受体相关因子6（TRAF6）的含量。分别应用酶联免疫吸附测定（ELISA）、细胞计数试剂盒-8（CCK8）、5-乙炔基-2'-脱氧尿苷（EdU）和流式细胞术分析炎症因子分泌、细胞增殖和凋亡情况。蛋白质免疫印迹法还分析了核因子κB（NF-κB）通路关键分子RELA原癌基因（P65）和IkappaBalpha（IκBα）的磷酸化水平。通过荧光素酶报告系统和RNA免疫沉淀（RIP）实验验证miR-940与circ_0099630或TRAF6之间的关系。
结果
在牙周炎中观察到circ_0099630和TRAF6丰度较高而miR-940表达较低，并且circ_0099630敲低减轻了脂多糖（LPS）诱导的人牙周膜细胞（PDLCs）损伤。证实了miR-940与circ_0099630或TRAF6之间的关系，而miR-940下调减弱了si-circ_0099630对LPS过表达诱导的PDLCs损伤的修复作用。同样，TRAF6上调削弱了miR-940过表达对LPS诱导的PDLCs损伤的缓解作用。circ_0099630沉默明显抑制了P65和IκBα的磷酸化水平，从而通过作用于miR-940/TRAF6轴减轻炎症反应。
结论
在牙周炎中，沉默circ_0099630通过靶向miR-940/TRAF6/NF-κB减轻LPS诱导的牙周膜细胞损伤。
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