Figure 6.

MicroRNA-342-3p targets Chi3l1 and inhibits endothelial inflammation and VSMC migration and proliferation. (A) The expression of miR-342-3p was determined by qPCR using endothelial-enriched RNA obtained from the LCA and RCA following partial carotid ligation in APPsw-Tg or non-Tg mice at 2 days post-ligation (n = 5 each, data are shown as mean ± SEM, *p < 0.05 as determined by Student's t-test). (B) iMAECs were treated with IL-6 (10 ng/mL; 24 h), then miR-342-3p expression was determined (n = 5, data shown as mean ± SEM, *p < 0.05 as determined by Student's t-test). Panel C shows the seed sequence of miR-342-3p and complementary 3'-UTR sequences of Chi3l1 wild-type and mutant. (D) iMAECs transfected with dual-luciferase reporter plasmids containing wild-type or mutant Chi3l1-3'-UTR were treated with miR-342-3p mimic or control mimic (Control-miR). Firefly luciferase activity (normalized to control Renilla luciferase) indicating Chi3l1 expression was determined using Luc-Pair miR Luciferase Assay Kit (n = 4 each, data shown as mean ± SEM, *p < 0.05 as determined by Student's t-test). (E-F) The expression of Chi3l1 (E) mRNA (n = 5, data are shown as mean ± SEM, *p < 0.05 as determined by Student's t-test) and (F) protein in IL-6-stimulated iMAECs in the presence of miR-342-3p mimic (50 nM) or control-miR were determined. (G-H) HUVECs and iMAECs were transfected with miR-342-3p (50 nM) for 24 h, then (G) THP1 monocyte adhesion to ECs and (H) NO level was determined (n = 5). (I) VSMCs were transfected with miR-342-3p (50 nM) or control-miR in serum-free medium for 24 h, then treated with PDGF-BB (25 ng/mL; 24 h), and cell migration (n = 3) and cell proliferation (n = 5) were determined.