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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：环状RNA（circularRNA，circRNA）是一种新型的功能性非编码RNA（ncRNA），在多种人类肝病的发生和发展中起作用，包括肝纤维化（LF）。LF是肝损伤后的可逆修复反应，肝星状细胞（HSC）的激活是核心事件。然而,circRNAs诱导LF中HSC活化的调控机制仍知之甚少。在这项研究中，研究了介导炎症反应并导致HSC活化的circAno6/miR-296-3p/toll样受体4（TLR4）信号轴。
方法：首先，将circAno6过表达质粒和小干扰RNA转染到细胞中以确定circAno6是否可以影响HSC的功能。第二,实时定量聚合酶链反应（RT-qPCR），酶联免疫吸附测定(ELISA),免疫印迹(WB)和免疫荧光(IF)检测circAno6质粒/siRNA转染对HSC活化指标的影响,炎症标志物和circAno6/miR-296-3p/TLR4信号轴。然后通过核质分离和荧光原位杂交（FISH）检查circAno6的亚细胞位置。最后,荧光素酶报告基因检测用于鉴定circAno6和miR-296-3p之间的关系,以及miR-296-3p和TLR4之间的关系.
结果：CircAno6在HSC中显著上调，并与细胞增殖和α-平滑肌肌动蛋白（α-SMA）呈正相关，胶原蛋白I，NOD-类受体热蛋白吲哚相关蛋白3（NLRP3），白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的表达。circAno6的过表达增加了炎症反应并诱导了HSC活化,而干扰导致相反的效果。FISH实验揭示了circAno6在细胞质中的定位。然后，双荧光素酶报告基因试验证实miR-296-3p显著抑制了circAno6-WT和TLR4-WT组的荧光素酶活性.
结论：这项研究表明circAno6和miR-296-3p/TLR4可能形成调节轴并调节炎症反应，这又诱导HSC激活。靶向cirAno6可能是治疗LF的潜在治疗策略。
方法：首先，将circAno6过表达质粒和小干扰RNA转染到细胞中以确定circAno6是否可以影响HSC的功能。第二,实时定量聚合酶链反应（RT-qPCR），酶联免疫吸附测定(ELISA),免疫印迹(WB)和免疫荧光(IF)检测circAno6质粒/siRNA转染对HSC活化指标的影响,炎症标志物和circAno6/miR-296-3p/TLR4信号轴。然后通过核质分离和荧光原位杂交（FISH）检查circAno6的亚细胞位置。最后,荧光素酶报告基因检测用于鉴定circAno6和miR-296-3p之间的关系,以及miR-296-3p和TLR4之间的关系.
结果：CircAno6在HSC中显著上调，并与细胞增殖和α-平滑肌肌动蛋白（α-SMA）呈正相关，胶原蛋白I，NOD-类受体热蛋白吲哚相关蛋白3（NLRP3），白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的表达。circAno6的过表达增加了炎症反应并诱导了HSC活化,而干扰导致相反的效果。FISH实验揭示了circAno6在细胞质中的定位。然后，双荧光素酶报告基因试验证实miR-296-3p显著抑制了circAno6-WT和TLR4-WT组的荧光素酶活性.
结论：这项研究表明circAno6和miR-296-3p/TLR4可能形成调节轴并调节炎症反应，这又诱导HSC激活。靶向cirAno6可能是治疗LF的潜在治疗策略。
