Supplementary Material for: Hsa Circ 001839 Promoted Inflammation in Renal Ischemia-Reperfusion Injury Through NLRP3 by miR-432-3p

<b><i>Background:</i></b> In recent years, increasing discovery of the extremely important regulatory effects of circular RNAs on biological development, angiogenesis, tumor genesis, and development, as well as stem cell proliferation and differentiation has provided new opportunities for investigating regulation mechanism in angiogenesis. <b><i>Objectives:</i></b> This study explored the expression of circ 001839 in renal ischemia-reperfusion injury (RI-RI) rats and whether its upstream microRNA-432-3p (miR-432-3p) affects inflammation in both RI-RI rats and NRK52E cells. <b><i>Methods:</i></b> Rat model of RI-RI was made, and circ 001839 was identified by the gene-chip analysis in RI-RI rats. Expression of circ 001839 and miR-432-3p was measured by reverse transcription-quantitative polymerase chain reaction, protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, IL-6, and IL-18 in rat serum and cell supernatant was determined by ELISA, and the expression of NOD-like receptor 3 (NLRP3) and other gap-associated proteins in NRK52E cells was evaluated by Western blot analysis. Next, to verify the regulatory relationship between circ 001839 and miR-432-3p, 2 luciferase reporters were constructed. <b><i>Results:</i></b> Circ 001839 expression of RI-RI rats and NRK52E cells was significantly upregulated, compared with the control group. Circ 001839 overexpression significantly increased inflammation through promoting TNF-α, IFN-γ, and IL-6 expression levels in NRK52E cells. Overexpression of miR-432-3p significantly promoted inflammation in NRK52E cells via induction of NLRP3. Moreover, miR-432-3p decreased the effects of circ 001839-induced inflammation in NRK52E cells. <b><i>Conclusions:</i></b> These findings suggested that circ 001839 promoted inflammation in RI-RI through NLRP3 by miR-432-3p.

**研究背景：** 近年来，环状RNA（circular RNA）在生物发育、血管生成、肿瘤发生发展以及干细胞增殖分化中的重要调控作用被不断揭示，这为探究血管生成的调控机制提供了新的契机。
**研究目的：** 本研究旨在探究环状RNA circ001839在肾缺血再灌注损伤（renal ischemia-reperfusion injury, RI-RI）大鼠中的表达情况，以及其上游微小RNA-432-3p（microRNA-432-3p, miR-432-3p）是否会对RI-RI大鼠与NRK52E细胞中的炎症反应产生影响。
**研究方法：** 构建肾缺血再灌注损伤大鼠模型，通过基因芯片分析鉴定该模型大鼠体内的circ001839。采用逆转录定量聚合酶链反应（reverse transcription-quantitative polymerase chain reaction, RT-qPCR）检测circ001839与miR-432-3p的表达水平；通过酶联免疫吸附测定（enzyme linked immunosorbent assay, ELISA）检测大鼠血清及细胞上清液中肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素-1β（interleukin-1β, IL-1β）、干扰素-γ（interferon-γ, IFN-γ）、IL-6及IL-18的蛋白表达水平；采用蛋白质印迹法（Western blot analysis）检测NRK52E细胞中NOD样受体3（NOD-like receptor 3, NLRP3）及其他间隙相关蛋白的表达。为验证circ001839与miR-432-3p的调控关系，本研究构建了2个荧光素酶报告基因载体。
**研究结果：** 与对照组相比，肾缺血再灌注损伤大鼠及NRK52E细胞中circ001839的表达水平显著上调。在NRK52E细胞中，circ001839过表达可通过上调TNF-α、IFN-γ及IL-6的表达水平，显著加剧炎症反应。miR-432-3p过表达可通过诱导NLRP3的表达，显著促进NRK52E细胞的炎症反应。此外，miR-432-3p可削弱circ001839诱导的NRK52E细胞炎症效应。
**研究结论：** 上述研究结果表明，circ001839可通过miR-432-3p介导NLRP3通路，进而促进肾缺血再灌注损伤中的炎症反应。