Experimental Center of Clinical Research, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui Province 230031, China; School of Pharmacy, Anhui University of Chinese Medicine, Hefei, Anhui Province 230012, China.
Experimental Center of Clinical Research, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui Province 230031, China.
Gene. 2024 Aug 20;920:148497. doi: 10.1016/j.gene.2024.148497. Epub 2024 Apr 26.
Circular RNA (circRNA) is a novel functional non-coding RNA(ncRNA) that plays a role in the occurrence and development of multiple human liver diseases, including liver fibrosis (LF). LF is a reversible repair response after liver injury, and the activation of hepatic stellate cells (HSCs) is the core event. However, the regulatory mechanisms by which circRNAs induce the activation of HSCs in LF are still poorly understood. The circAno6/miR-296-3p/toll-like receptor 4 (TLR4) signaling axis that mediates the inflammatory response and causes the activation of HSCs was investigated in this study.
First, a circAno6 overexpression plasmid and small interfering RNA were transfected into cells to determine whether circAno6 can affect the function of HSCs. Second, real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting (WB) and immunofluorescence (IF) were used to detect the effects of circAno6 plasmid/siRNA transfection on HSC activation indices, inflammatory markers and the circAno6/miR-296-3p/TLR4 signaling axis. The subcellular position of circAno6 was then examined by nucleo-cytoplasmic separation and fluorescence in situ hybridization (FISH). Finally, a luciferase reporter gene assay was used to identify the relationship between circAno6 and miR-296-3p as well as the relationship between miR-296-3p and TLR4.
CircAno6 was considerably upregulated in HSCs and positively correlated with cell proliferation and alpha-smooth muscle actin (α-SMA), collagen I, NOD-likereceptorthermalproteindomainassociatedprotein 3 (NLRP3), interleukin-1β (IL-1β) and interleukin-18 (IL-18) expression. Overexpression of circAno6 increased the inflammatory response and induced HSC activation, whereas interference resulted in the opposite effects. FISH experiments revealed the localization of circAno6 in the cytoplasm. Then, a double luciferase reporter assay confirmed that miR-296-3p significantly inhibited luciferase activity in the circAno6-WT and TLR4-WT groups.
This study suggests that circAno6 and miR-296-3p/TLR4 may form a regulatory axis and regulate the inflammatory response, which in turn induces HSC activation. Targeting circAno6 may be a potential therapeutic strategy to treat LF.
环状 RNA（circRNA）是一种新型的功能非编码 RNA（ncRNA），在多种人类肝脏疾病的发生和发展中发挥作用，包括肝纤维化（LF）。LF 是肝损伤后一种可逆的修复反应，肝星状细胞（HSCs）的激活是核心事件。然而，circRNAs 诱导 LF 中 HSCs 激活的调节机制仍知之甚少。本研究探讨了介导炎症反应并导致 HSCs 激活的 circAno6/miR-296-3p/ toll 样受体 4（TLR4）信号轴。
首先，转染 circAno6 过表达质粒和小干扰 RNA 以确定 circAno6 是否可以影响 HSCs 的功能。其次，通过实时定量聚合酶链反应（RT-qPCR）、酶联免疫吸附测定（ELISA）、Western blot（WB）和免疫荧光（IF）检测 circAno6 质粒/siRNA 转染对 HSC 激活指标、炎症标志物和 circAno6/miR-296-3p/TLR4 信号轴的影响。然后通过核质分离和荧光原位杂交（FISH）检测 circAno6 的亚细胞位置。最后，通过荧光素酶报告基因检测鉴定 circAno6 与 miR-296-3p 以及 miR-296-3p 与 TLR4 之间的关系。
circAno6 在 HSCs 中显著上调，与细胞增殖和α-平滑肌肌动蛋白（α-SMA）、胶原 I、NOD 样受体热蛋白结构域相关蛋白 3（NLRP3）、白细胞介素 1β（IL-1β）和白细胞介素 18（IL-18）的表达呈正相关。circAno6 的过表达增加了炎症反应并诱导 HSC 激活，而干扰则产生相反的效果。FISH 实验显示 circAno6 定位于细胞质中。然后，双荧光素酶报告基因检测证实 miR-296-3p 显著抑制 circAno6-WT 和 TLR4-WT 组的荧光素酶活性。
本研究表明，circAno6 和 miR-296-3p/TLR4 可能形成一个调节轴，调节炎症反应，进而诱导 HSC 激活。靶向 circAno6 可能是治疗 LF 的一种潜在治疗策略。
