NF-κB-Regulated miR-99a Modulates Endothelial Cell Inflammation

Abstract

Objective. The present study was performed to investigate the effects and mechanisms of miR-99a on LPS-induced endothelial cell inflammation, as well as the regulation of NF-κB on miR-99a production. Methods and Results. ELISA showed that LPS treatment significantly promoted the secretion of inflammatory factors (TNF-α, IL-6, IL-1β, and MCP-1). LPS treatment also inhibited miR-99a production and promoted mTOR expression and NF-κB nuclear translocation. Overexpression of miR-99a suppressed the LPS-induced TNF-α, IL-6, IL-1β, and MCP-1 overproduction, mTOR upregulation, and NF-κB nuclear translocation. The PROMO software analysis indicated NF-κB binding site in the -1643 to -1652 region of miR-99a promoter. Dual luciferase reporter analysis, electrophoretic mobility shift assays (EMSA), and chromosome immunoprecipitation (ChIP) assays demonstrated that NF-κB promoted the transcription of miR-99a by binding to the -1643 to -1652 region of miR-99a promoter. Further studies on HUVECs verified the regulatory effects of NF-κB on miR-99a production. Conclusion. MiR-99a inhibited the LPS-induced HUVECs inflammation via inhibition of the mTOR/NF-κB signal. NF-κB promoted miR-99a production by binding to the -1643 to -1652 region of miR-99a promoter. Considering the importance of endothelial inflammation on cardiovascular diseases, such as atherosclerosis, our results may provide a new insight into the pathogenesis and therapy of atherosclerosis.

Figure 1

Effects of LPS on miR-99a production and effects of miR-99a on LPS-induced inflammation in HUVECs. (a) Effects of LPS treatment at different concentrations for 24 hours on miR-99a expression; (b) effects of different time treatment with LPS (20 μg/mL) on miR-99a expression; (c) effects of miR-99a on LPS-induced inflammatory factors secretion. The values (mean ± SD from three independent experiments). ^# P < 0.05; ^## P < 0.01 versus control; ^∗ P < 0.05; ^∗∗ P < 0.01 versus LPS group; ^∗∗∗ P < 0.001 versus LPS group.

Figure 2

Effects of miR-99a on LPS-induced mTOR expression and NF-κB nuclear translocation. Endothelial cells were transfected with miR-99a (100 nM) mimic or rapamycin (50 nM) for 24 hours and then treated with LPS (20 μg/mL) for another 24 hours. The protein levels were detected by Western-blot. (a) The expression of different proteins analyzed by Western-blot; (b) quantified band density for mTOR relative to control, which was set as 1; (c) percentage of NF-κB in nucleus, total NF-κB presented by the summary of quantified band density for NF-κB (cytoplasm) and NF-κB (nucleus). All values are presented as mean ± SD from three independent experiments. ^# P < 0.05; ^## P < 0.01 versus control; ^∗ P < 0.05; ^∗∗ P < 0.01 versus LPS.

Figure 3

Regulatory effects of NF-κB on miR-99a expression in HUVECs. ((a), (b)) Relative miR-99a expression in HUVECs after NF-κB overexpression or inhibition. ((c), (d)) The increased p50 or p65 protein levels in HUVECs which were transfected with 4 μg of NF-κB subunit ORF cDNA Clones (p50, p65, or p50 + p65) in 6-well plate for 48 hours. The values (mean ± SD from three independent experiments) are relative to NC or control, which was set as 1. ^∗ P < 0.05; ^∗∗ P < 0.01 versus NC group.

Figure 4

Dual luciferase reporter analysis of the regulate effects of NF-κB on miR-99a promoter plasmids. (a) The predicted NF-κB binding sites in miR-99a promoter analyzed by PROMO software. ((b), (c)) Portions of the upstream region of miR-99a cloned to create −1.5 kb, −2.0 kb, and −2.0 kb-Mut plasmids. (d) The increased p65 or p50 protein levels in HEK293 cells after being transfected with 150 ng of NF-κB subunit ORF cDNA Clones (p50, p65, or p50 + p65) in 24-well plates for 48 hours. (e) Relative luciferase activity of different promoter reporter plasmids after NF-κB overexpression. The values (mean ± SD from three independent experiments) are relative to NC-Blank, which was set as 1. ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 5

EMSA and ChIP verification of interactions of NF-κB with its binding sites in the miR-99a promoter. (a) EMSA validation of NF-κB binding sites on miR-99a promoter. Lane 1, labeled positive NF-κB probe only; lane 2, nuclear extracts plus labeled positive NF-κB probe; lane 3, labeled WT probe only; lane 4, nuclear extracts plus labeled WT probe; lanes 5 and 6, nuclear extracts plus labeled WT probe and cold unlabeled WT probe (50-fold and 100-fold labeled WT probe), respectively; lanes 7 and 8, nuclear extracts plus labeled WT probe and cold unlabeled Mut probe (50-fold and 100-fold labeled WT probe), respectively. NC: negative control; PC: positive control; NE: nuclear extracts; WT: wild-type probe; NWT: nonlabeled wild-type probe; NMut: nonlabeled mutant probe; P: free biotin-labeled probe. (b) Representative gel images of ChIP assay. (c) Relative NF-κB binding activity quantified by the ChIP bands. ^∗ P < 0.05 versus control group.