Fig. 3: HSPA7 promotes inflammatory changes in HASMCs by sponging miR-223. | Experimental & Molecular Medicine

Fig. 3: HSPA7 promotes inflammatory changes in HASMCs by sponging miR-223.

From: LncRNA HSPA7 in human atherosclerotic plaques sponges miR-223 and promotes the proinflammatory vascular smooth muscle cell transition

Fig. 3

a Bioinformatics analysis using miRcode predicted that HSPA7 contains a potential miR-223 binding site. miRDB revealed that FOXO1 was a target of miR-223. b After transfection of HASMCs with miR-223, qPCR showed significantly downregulated miR-223 expression. c HASMCs were transfected with a miR-223 inhibitor and/or siHSPA7 or control siRNA and then treated with oxLDL. The secretion of IL-1β and IL-6 was increased upon miR-223 inhibitor treatment, whereas it was diminished after siHSPA7 treatment, particularly for IL-6. These findings were verified using qPCR. d After the same treatment of HASMCs, qPCR showed that upregulated expression of FOXO1 induced by the miR-223 inhibitor was diminished after siHSPA7 treatment. Although luciferase-reported NF-κB activity was elevated upon miR-223 inhibition, this parameter was attenuated by siHSPA7. e After the same treatment of THP-1 cell-derived macrophages, the secretion of IL-1β and IL-6 was measured and showed a similar tendency to that in HASMCs. qPCR demonstrated up- and downregulated expression of the corresponding genes and FOXO1. f Immunofluorescence staining showed that the expression of markers of the contractile SMC phenotype did not change upon miR-223 inhibitor treatment; however, they increased after siHSPA7 treatment. g These findings were verified using qPCR for TAGLN and CNN1. h miR-223 is a target of HSPA7 in an AGO2-dependent manner. AGO2 expression was not different in HASMCs regardless of the presence of oxLDL. RIP assays showed that HSPA7 and miR-223 were enriched in AGO2-containing miRNPs compared to IgG immunoprecipitates. HSPA7 had increased binding to AGO2 in the presence of oxLDL. HASMCs were transfected with or without miR-223 inhibitor and then treated with oxLDL. HAPA7 expression was shown to be significantly lower in AGO2 immunoprecipitates in the cells treated with the miR-223 inhibitor. Data are from at least three independent experiments. *: p < 0.05. HASMC human aortic smooth muscle cell; oxLDL oxidized low-density lipoprotein; C control; AGO argonaute-2; RIP RNA immunoprecipitation; miRNP miRNA ribonucleoprotein.

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