Site-specific microRNA-92a regulation of Kruppel-like factors 4 and 2 in atherosusceptible endothelium

Abstract

Objective: Endothelial transcription factors Krüppel-like factor 4 (KLF4) and KLF2 are implicated in protection against atherogenesis. Steady-state microRNA (miR) regulation of KLFs in vivo is accessible by screening region-specific endothelial miRs and their targets.

Methods and results: A subset of differentially expressed endothelial miRs was identified in atherosusceptible versus protected regions of normal swine aorta. In silico analyses predicted highly conserved binding sites in the 3'-untranslated region (3'UTR) of KLF4 for 5 miRs of the subset (miR-26a, -26b, -29a, -92a, and -103) and a single binding site for a miR-92a complex in the 3'UTR of KLF2. Of these, only miR-92a knockdown and knock-in resulted in responses of KLF4 and KLF2 expression in human arterial endothelial cells. Dual luciferase reporter assays demonstrated functional interactions of miR-92a with full-length 3'UTR sequences of both KLFs and with the specific binding elements therein. Two evolutionarily conserved miR-92a sites in KLF4 3'UTR and 1 site in KLF2 3'UTR were functionally validated. Knockdown of miR-92a in vitro resulted in partial rescue from cytokine-induced proinflammatory marker expression (monocyte chemotactic protein 1, vascular cell adhesion molecule-1, E-selectin, and endothelial nitric oxide synthase) that was attributable to enhanced KLF4 expression. Leukocyte-human arterial endothelial cell adhesion experiments supported this conclusion. In swine aortic arch endothelium, a site of atherosusceptibility where miR-92a expression was elevated, both KLFs were expressed at low levels relative to protected thoracic aorta.

Conclusions: miR-92a coregulates KLF4 and KLF2 expression in arterial endothelium and contributes to phenotype heterogeneity associated with regional atherosusceptibility and protection in vivo.

Figure 1

miR-92a regulation of endothelial KLF4 and KLF2. A-D: Knock-down of miRs (A) selectively influenced expression of KLF4/KLF2 mRNA (B, C) and protein (D) in HAECs (n=4-6). E, F: miR-92a knock-in suppressed expression of KLF4 and KLF2 in HAECs (n=4). Data represent mean ± SEM. *p < 0.05. KD: knock-down; Kin: knock-in.

Figure 2

miR-92a targeting of the 3′ UTRs of KLF4 and KLF2. A: Evolutionarily-conserved putative miR-92a binding sites in 3′ UTRs of KLF4 and KLF2. B: Reduced luciferase activity in HEK293 cells overexpressing miR-92a mimetics or miR-92a precursors following insertion of the full-length 3′UTRs cloned from human KLF4 and KLF2 (n=4-5). Ctl plasmids express wild-type firefly luciferase without 3′ UTR insertions. Data represent mean ± SEM. *p < 0.05.

Figure 3

miR-92a negatively regulates KLF4 and KLF2 through the seed-pairing sequence(s) in the 3′UTR binding site(s). A: Schematic representation of the luciferase vectors containing the full-length 3′ UTR(s) of KLF4 and KLF2 and mutation(s) in the miR-92a seed-pairing site(s). B: Reduced luciferase activity in HEK293 cells overexpressing miR-92a precursors following insertion of the mutant clones (n=3-5). Data represent mean ± SEM. *p < 0.05.

Figure 4

miR-92a negatively regulates KLF4 and KLF2 through the miR-92a recognition elements in the 3′UTR binding site(s). A: Schematic representation of the luciferase vectors containing the corresponding miR-92a binding sites. B: Reduced luciferase activity in HEK293 cells overexpressing miR-92a mimetics following insertion of miR-92a putative binding elements cloned from KLF4 and KLF2 3′ UTRs. Renilla luciferase inserted with putative let-7b binding sites, cloned from LIN-28 3′ UTR, served as negative control (n=3-5). *p < 0.05. C-E: Seed sequence-mediated regulation of KLF4 by miR-92a. C: Point mutations in the miR-92a binding elements that disrupt the interaction between miR-92a seed region and the 3′ UTR of KLF4. D: Schematic representation of the luciferase vectors containing the combinations of wild-type/mutated conserved and less-conserved miR-92a binding sites cloned from 3′ UTR of KLF4. E: Reduced luciferase activity in HEK293 cells overexpressing miR-92a following insertion of miR-92a binding elements cloned from wild-type conserved and less-conserved miR-92a binding sites (n=3-5). Data represent mean ± SEM. *p < 0.05.

Figure 4

miR-92a negatively regulates KLF4 and KLF2 through the miR-92a recognition elements in the 3′UTR binding site(s). A: Schematic representation of the luciferase vectors containing the corresponding miR-92a binding sites. B: Reduced luciferase activity in HEK293 cells overexpressing miR-92a mimetics following insertion of miR-92a putative binding elements cloned from KLF4 and KLF2 3′ UTRs. Renilla luciferase inserted with putative let-7b binding sites, cloned from LIN-28 3′ UTR, served as negative control (n=3-5). *p < 0.05. C-E: Seed sequence-mediated regulation of KLF4 by miR-92a. C: Point mutations in the miR-92a binding elements that disrupt the interaction between miR-92a seed region and the 3′ UTR of KLF4. D: Schematic representation of the luciferase vectors containing the combinations of wild-type/mutated conserved and less-conserved miR-92a binding sites cloned from 3′ UTR of KLF4. E: Reduced luciferase activity in HEK293 cells overexpressing miR-92a following insertion of miR-92a binding elements cloned from wild-type conserved and less-conserved miR-92a binding sites (n=3-5). Data represent mean ± SEM. *p < 0.05.

Figure 5

miR-92a primes endothelial inflammation by inhibiting athero-protective KLF4. A, B, C: Knockdown of endogenous miR-92a desensitizes the TNFα-induced inflammatory markers MCP-1 (A), VCAM-1 (B) and E-SEL (C). The rescue effect was reversed in cells in which both miR-92a and KLF4 (siRNA) were knocked-down (n=4). D: Rescue of TNFα-induced inhibition of eNOS in endothelial cells knockdown of endogenous miR-92a and reversal following knockdown of both miR-92a and KLF4 (n=4). E, F: Knockdown of endogenous miR-92a in HAECs decreases THP-1 cell adhesion to HAEC while simultaneous siRNA inhibition of KLF4 restores the level of leukocyte-endothelial interaction. Representative images of fluorescence-labeled THP-1 cells (E), and quantitative fluorescence measurements (F). n=4. Data represent mean ± SEM. *p < 0.05.

Figure 6

Endothelial expression of miR-92a, KLF4, and KLF2 in swine athero-susceptible aortic arch (AA) and athero-resistant descending thoracic aorta (DT). A: Low expression of endothelial miR-92a in DT referenced to AA (n=7-9). B, C: Increased expression of endothelial KLF4 and KLF2 mRNA (B) and protein (C) in DT aorta referenced to AA (n=7). Data represent mean ± SEM. *p < 0.05