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.
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。