The circAno6/miR-296-3p/TLR4 signaling axis mediates the inflammatory response to induce the activation of hepatic stellate cells

Zhen Li; Yanzhen Ma; Chang Fan; Hui Jiang

doi:10.1016/j.gene.2024.148497

The circAno6/miR-296-3p/TLR4 signaling axis mediates the inflammatory response to induce the activation of hepatic stellate cells

Gene. 2024 Apr 26:920:148497. doi: 10.1016/j.gene.2024.148497. Online ahead of print.

Authors

Zhen Li¹, Yanzhen Ma¹, Chang Fan², Hui Jiang³

Affiliations

¹ 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.
² Experimental Center of Clinical Research, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui Province 230031, China.
³ 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. Electronic address: jianghui@ahtcm.edu.cn.

PMID: 38677350
DOI: 10.1016/j.gene.2024.148497

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: HSCs; Inflammatory response; Liver fibrosis; circAno6/miR-296-3p/TLR4 signaling axis.