Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells. (2020)
Figure 1

Figure 1. The novel miR-3679-5p suppresses monocyte adhesion and targets two histone demethylases, KDM7A and UTX, in human inflammatory activated ECs.

(A) Heat map representation of the TNF-α-up-regulated genes. Genes were classified based on the TNF-α-treated time points, using the algorithm "HOPACH".

(B) Heat map showing down-regulated miRNAs at 4 hrs, but not 24 hrs, after TNF-α treatment. The middle column indicates the fold-change values (control/TNF) at 4 hrs. The right column indicates the relative expression level of each miRNA from the FANTOM5 database (Consortium et al, 2014). Asterisks indicate the top three most down-regulated miRNAs which were reported to be expressed in human endothelial cells (ECs) according to the FANTOM5 database.

(C) Bar plots showing mean mRNA levels of vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1), and E-selectin (SELE), measured 4 hrs after stimulation of human ECs with or without TNF-α ± miR-3679-5p. Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 and ‡ P = 0.203 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.

(D) Western blots for VCAM1, ICAM, SELE, and β-actin (ACTB) in lysates from ECs treated with or without TNF-α (4 hrs) ± miR-3679-5p.

(E) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± miR-3679-5p. Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.

(F) The scatter plot shows the correlation of RNA immunoprecipitation (RIP)　followed by microarray data from two replicate screens for the identification of miR-3679-5p target genes. Genes up- and down-regulated by miR-3679-5p treatment (|average Log2FC|>1) are indicated in red and blue, respectively.

(G) Venn diagram showing the overlap of the genes selected by RIP-ChIP (1,334) and TargetScan7.0 (1,224) (http://www.targetscan.org/vert_70/).

(H) Gene ontology (GO) analysis of the 253 potential target genes for miR-3679-5p, as determined in Fig 1G. The P-values of each category analyzed from DAVID (https://david.ncifcrf.gov/) are shown in the bar graphs.

(I) Seed sequence of miR-3679-5p and complementary 3′ UTR sequence of lysine demethylase 7A (KDM7A). The letters in red indicate matched bases.

(J and K) Effect of miR-3679-5p mimics (J) or miR-3679-5p inhibitor (K) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of KDM7A. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.

(L) Seed sequence of miR-3679-5p and complementary 3′ UTR sequence of lysine demethylase 6A (UTX). The letters in red indicate matched bases.

(M and N) The effect of miR-3679-5p mimics (M) or miR-3679-5p inhibitor (N) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of UTX was measured. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.