Fig 1.
Effects of miR-27a/b on LPL expression and activities in various cell types.
(A and B) LPL mRNA and protein levels were measured by RT-qPCR and western blot assay, respectively, in THP-1 cells transfected with miR-27 mimic or inhibitor. (C and D) LPL mRNA and protein levels were measured by RT-qPCR and western blot assay in RAW 264.7 cells transfected with miR-27 mimic or inhibitor, respectively. (E) Changes of LPL activities in the culture media of THP-1 macrophages and RAW 264.7 cells treated with miR-27a/b mimic or inhibitor, respectively. All results are expressed as mean±S.D. from three independent experiments. *P<0.05 vs. mimic-neg, #P<0.05 vs. inhibitor-neg.
Fig 2.
Effects of miR-27a/b on LPL expression in apoE KO mice.
Eight-week-old male apoE KO mice (n = 10 mice per group) fed high-fat diet were given a tail vein injection with miR-27a/b agomir (AGa and AGb) or its scrambled agomir negative control (AG-NC), miR-27a/b antagomir (ANa and ANb) or its scrambled antagomir negative control (AN-NC). (A and B) LPL mRNA and protein levels in aortic tissues of apoE KO mice were measured by RT-qPCR, respectively. (C and D) LPL mRNA and protein levels in peritoneal macrophages of apoE KO mice were measured by RT-qPCR and western blot assay, respectively. All results are expressed as mean±S.D. *P<0.05 vs. AG-NC, #P<0.05 vs. AN-NC.
Table 1.
Effects of miR-27a/b on secretion of proinflammatory cytokines in ox-LDL- treated THP-1 macrophages.
Fig 3.
Effects of miR-27a/b on the expression of NF-κB in human THP-1 macrophages.
Expression of NF-κB protein and its phosphorylation status was detected by western blot analysis in THP-1 macrophages treated with miR-27a/b mimic or inhibitor.
Table 2.
Effects of miR-27a/b on inflammatory cytokine production in the blood of apoE KO mice.
Fig 4.
Effects of miR-27a/b on the expression of surface scavenger receptors.
THP-1 macrophages were incubated with miR-27a/b mimic or inhibitor for 6 h, and then with ox-LDL for 24 h. SR-A1, LOX-1, CD36 and CXCL16 expression was determined after incubation with miR-27a/b mimic or inhibitor. (A) The relative mRNA levels of SR-A1, LOX-1, CD36 and CXCL16. (B and C) The protein levels of SR-A1, LOX-1, CD36 and CXCL16 were detected by western blot analysis. All results are expressed as mean±S.D. from three independent experiments. *P<0.05 vs mimic-neg, #P<0.05 vs inhibitor-neg, **P<0.01 vs mimic-neg, ##P<0.01 relative to inhibitor-neg.
Table 3.
Effects of miR-27a/b on lipid accumulation in macrophages in abdominal cavity of apoE KO mice.
Fig 5.
Effects of bovine LPL and LPL siRNA on the expressions and activities of LPL in ox-LDL-stimulated THP-1 macrophages.
Confirmation of siRNA-induced knockdown of LPL activities (A), mRNA (B) and protein levels (C) after transfection with LPL-siRNA or an irrelevant control sequence (siRNA-neg) or treatment with and without bovine LPL in ox-LDL-stimulated THP-1 macrophages. All results are expressed as mean±S.D. from three independent experiments. *P<0.05 vs control. #P<0.05 vs siRNA-neg.
Fig 6.
Effects of LPL on miR-27a/b-regulated expression of surface scavenger receptors in ox-LDL-stimulated THP-1 macrophages.
The mRNA levels of SR-A1, LOX1, CD36 and CXCL16 were measured by RT-qPCR in THP-1 macrophages transfected with LPL siRNAs and then incubated with miR-27a/b inhibitors, respectively. All results are expressed as mean±S.D. from three independent experiments. *P<0.05 vs miR-27a inhibitor. #P<0.05 vs miR-27b inhibitor.
Table 4.
The Effects of miR-27a/b on body weight and plasma lipid profile in apoE KO mice.
Fig 7.
MiR-27a/b inhibits aortic atherosclerosis.
Male 8-week-old apoE KO mice (n = 10 mice per group) fed high fat diet were given a tail vein injection with miR-27a/b agomir negative control (AG-NC), miR-27a/b agomir (AGa/AGb), miR-27a/b antagomir negative control (AN-NC) and miR-27a/b antagomir (ANa/ANb). (A) MiR-27a/b alleviates atherosclerotic plaque development in apoE KO mice. Plaques (arrows) in aortic arches and thoracic aortas of representative apoE KO mice are shown. (B) Effects of miR-27a/b on atherosclerotic lesion areas in apoE KO mice. Representative images and the quantification of atherosclerotic lesion areas in the en face analysis of the whole aorta with Oil red O staining. (C) Effects of miR-27a/b on aortic atherosclerotic lesions in aortic root in apoE KO mice. Representative micrographs were obtained from hematoxylin-eosin staining of cross-sections of proximal aorta in apoE KO mice. (D) Effects of miR-27a/b on the aortic sinus lesion areas in apoE KO mice. Characterization of aortic sinus atherosclerotic lesion areas was performed by Oil red O staining. Total Oil red O staining positive area was determined using IMAGEPRO PLUS Software. Images of representative sections from each group are accompanied by summarized bar charts. Each data point represents an individual animal. The horizontal lines denote the mean of each group. All results are expressed as mean±S.D.*P<0.05 vs AG-NC. #P<0.01 vs AN-NC.
Fig 8.
A role for miR-27 in negative regulation of lipid accumulation and proinflammatory cytokine secretion through targeting LPL gene.
MiR-27, binding to the LPL 3’UTR to accelerate degradation or repress post-transcriptional expression of mRNA, can inhibit the expression of LPL, and then attenuate lipid accumulation and secretion of proinflammatory cytokines.