Fig. 8

Short-term activated HSC-sEV-specific miR-99a-5p might influence macrophage differentiation by targeting CD93. (A) Dual-luciferase assay for the interaction between miR-99a-5p and the 3’UTR of CD93 mRNA in 293T cells. (B) The effects of the miR-99a-5p mimic on CD93 expression in primary rat liver macrophages (KCs) and human THP-1 macrophages were measured by RT-qPCR and western blotting. (C) The effects of agomir-99a-5p on CD93 expression in CCl4-induced chronic liver injury mice were detected by western blotting. (D) Spearman correlation between the expression of CD93 and CD206 in human HCC tissues; correlation p value = 1.41e-21, correlation coefficient = 0.47. (E) The mRNA expression of genes associated with HSC-sEV-induced macrophage differentiation in siCD93 treated human THP-1 macrophages was determined by RT-qPCR. (F) The mRNA expression of genes associated with HSC-sEV-induced macrophage differentiation in OE-CD93 treated human THP-1 macrophages was determined by RT-qPCR. (G, H) Cytokine array analyses for culture media from human THP-1 macrophages (G) or primary KCs (H) treated by siCD93, OE-CD93 and corresponding NC. Inflammatory factors are listed on the right side. Each row represents an individual inflammatory factor, and each column represents an individual sample. The scale represents normalized log2 inflammatory factor expression levels. Red font, proinflammatory factors; green font; anti-inflammatory factors. (I) The protein expression of TNF-α and CD206 in miR-99a-5p mimic treated CD93 overexpression human THP-1 macrophages were detected by western blotting. The relative protein expression was normalized to β-actin, and then to OE-NC. Statistical significance was determined by Student’s t test. Compared to OE-NC, ***p < 0.001, **p < 0.01, *p < 0.05; compared to OE-CD93, #p < 0.05. WT, wild type; MT, mutated type; NC, negative control