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Cited by 127MicroRNA in disease regulationAtherosclerosis and Cardiovascular DiseasesCytokine Signaling Pathways and Interactions

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miR-155 Regulated Inflammation Response by the SOCS1-STAT3-PDCD4 Axis in Atherogenesis

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Ye, Jinshan, Guo, Ruiwei, Shi, Yankun, Qi, Feng, Guo, Chuanming, Yang, Lixia·2016·Mediators of Inflammation
DOI 10.1155/2016/8060182PMC PMC5098093
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Abstract

Inflammation response plays a critical role in all phases of atherosclerosis (AS). Increased evidence has demonstrated that miR-155 mediates inflammatory mediators in macrophages to promote plaque formation and rupture. However, the precise mechanism of miR-155 remains unclear in AS. Here, we also found that miR-155 and PDCD4 were elevated in the aortic tissue of atherosclerotic mice and ox-LDL treated RAW264.7 cells. Further studies showed that miR-155 not only directly inhibited SOCS1 expression, but also increased the expression of p-STAT and PDCD4, as well as the production of proinflammation mediators IL-6 and TNF-α. Downregulation of miR-155 and PDCD4 and upregulation of SOCS1 obviously decreased the IL-6 and TNF-α expression. In addition, inhibition of miR-155 levels in atherosclerotic mice could notably reduce the IL-6 and TNF-α level in plasma and aortic tissue, accompanied with increased p-STAT3 and PDCD4 and decreased SOCS1. Thus, miR-155 might mediate the inflammation in AS via the SOCS1-STAT3-PDCD4 axis. These results provide a rationale for intervention of intracellular miR-155 as possible antiatherosclerotic targets.

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Figure 1 The relative expression of miR-155 in macrophages RAW264.7 cells and the aortic tissue of ApoE−/− mice. (a) qPCR analyzed the expression of miR-155 in RAW264.7 cells when macrophages were treated by ox-LDL for 24 h at the indicated dose: ∗ p < 0.05, relative to control, ∧ p < 0.05, relative to 5 μg/mL ox-LDL group, # p < 0.05, relative to 10 μg/mL ox-LDL group. (b) qPCR detected the level of miR-155 in macrophages, which were treated with 20 μg/mL ox-LDL at the indicated time: ∗ p < 0.05, relative to control, ∧ p < 0.05, relative to 6 h group, # p < 0.05, relative to 12 h group. (c) Expression of miR-155 in aortic tissue of ND-fed wild-type (WT) c57 mice, ND-fed ApoE−/− mice (ApoE−/− ND), and HFD-fed ApoE−/− mice (ApoE−/− HFD) was determined by qPCR (n = 4): ∗ p < 0.05, relative to c57 mice WT; ∧ p < 0.05, relative to ApoE−/− ND.
Figure 1 The relative expression of miR-155 in macrophages RAW264.7 cells and the aortic tissue of ApoE−/− mice. (a) qPCR analyzed the expression of miR-155 in RAW264.7 cells when macrophages were treated by ox-LDL for 24 h at the indicated dose: ∗ p < 0.05, relative to control, ∧ p < 0.05, relative to 5 μg/mL ox-LDL group, # p < 0.05, relative to 10 μg/mL ox-LDL group. (b) qPCR detected the level of miR-155 in macrophages, which were treated with 20 μg/mL ox-LDL at the indicated time: ∗ p < 0.05, relative to control, ∧ p < 0.05, relative to 6 h group, # p < 0.05, relative to 12 h group. (c) Expression of miR-155 in aortic tissue of ND-fed wild-type (WT) c57 mice, ND-fed ApoE−/− mice (ApoE−/− ND), and HFD-fed ApoE−/− mice (ApoE−/− HFD) was determined by qPCR (n = 4): ∗ p < 0.05, relative to c57 mice WT; ∧ p < 0.05, relative to ApoE−/− ND.CC BY · PubMed Central
Figure 2 The levels of cytokines IL-6, TNF-α, and IL-10 in RAW264.7 cells after ox-LDL treatment and atherosclerotic mice. (a) ox-LDL induced the expression of IL-6, TNF-α, and IL-10 for 24 h at indicated dose was determined by qPCR: ∗ p < 0.05, relative to control, ∧ p < 0.05, relative to 5 μg/mL ox-LDL group; # p < 0.05, relative to 10 μg/mL ox-LDL group. (b) The plasma levels of IL-6, TNF-α, and IL-10 of c57 mice WT, ApoE−/− ND, and ApoE−/− HFD were detected by the ELISA kit (n = 6): ∗ p < 0.05, relative to c57 mice WT; ∧ p < 0.05, relative to ApoE−/− ND.
Figure 2 The levels of cytokines IL-6, TNF-α, and IL-10 in RAW264.7 cells after ox-LDL treatment and atherosclerotic mice. (a) ox-LDL induced the expression of IL-6, TNF-α, and IL-10 for 24 h at indicated dose was determined by qPCR: ∗ p < 0.05, relative to control, ∧ p < 0.05, relative to 5 μg/mL ox-LDL group; # p < 0.05, relative to 10 μg/mL ox-LDL group. (b) The plasma levels of IL-6, TNF-α, and IL-10 of c57 mice WT, ApoE−/− ND, and ApoE−/− HFD were detected by the ELISA kit (n = 6): ∗ p < 0.05, relative to c57 mice WT; ∧ p < 0.05, relative to ApoE−/− ND.CC BY · PubMed Central
Figure 3 The relative expression of PDCD4 in macrophages RAW264.7 cells. (a and b) Western blot and qPCR analyzed the PDCD4 protein and mRNA expression after indicated ox-LDL treated macrophages RAW264.7 cells for 24 h. Representative bands show the protein expression of PDCD4 (the upper panel), β-actin (the middle panel), and the histograms showing quantification of PDCD4 bands normalization relative to β-actin expression in (a): ∗ p < 0.05 versus control, ∧ p < 0.05 versus 5 μg/mL group, and # p < 0.05 versus 10 μg/mL group. (c and d) Western blot and qPCR analyzed the PDCD4 protein and mRNA expression while RAW264.7 cells were treated by 20 μg/mL ox-LDL plus anti-miR-155 or anti-NC. 100 pM anti-miR-155 or anti-NC had been transfected into RAW264.7 cells for 24 h, and then they were treated by 20 μg/mL ox-LDL for 24 h. Representative bands show the protein expression of PDCD4 (the upper panel), β-actin (the middle panel), and the histograms showing quantification of PDCD4 bands normalized relative to β-actin expression in (c): ∗ p < 0.05 versus control, ∧ p < 0.05 versus 20 μg/mL ox-LDL group, and # p < 0.05 versus 20 μg/mL ox-LDL plus anti-NC group.
Figure 3 The relative expression of PDCD4 in macrophages RAW264.7 cells. (a and b) Western blot and qPCR analyzed the PDCD4 protein and mRNA expression after indicated ox-LDL treated macrophages RAW264.7 cells for 24 h. Representative bands show the protein expression of PDCD4 (the upper panel), β-actin (the middle panel), and the histograms showing quantification of PDCD4 bands normalization relative to β-actin expression in (a): ∗ p < 0.05 versus control, ∧ p < 0.05 versus 5 μg/mL group, and # p < 0.05 versus 10 μg/mL group. (c and d) Western blot and qPCR analyzed the PDCD4 protein and mRNA expression while RAW264.7 cells were treated by 20 μg/mL ox-LDL plus anti-miR-155 or anti-NC. 100 pM anti-miR-155 or anti-NC had been transfected into RAW264.7 cells for 24 h, and then they were treated by 20 μg/mL ox-LDL for 24 h. Representative bands show the protein expression of PDCD4 (the upper panel), β-actin (the middle panel), and the histograms showing quantification of PDCD4 bands normalized relative to β-actin expression in (c): ∗ p < 0.05 versus control, ∧ p < 0.05 versus 20 μg/mL ox-LDL group, and # p < 0.05 versus 20 μg/mL ox-LDL plus anti-NC group.CC BY · PubMed Central
Figure 4 Downregulation of PDCD4 and miR-155 regulated the expression of IL-6, TNF-α, and IL-10. (a) Knocking down the expression of PDCD4 mRNA by PDCD4 siRNA in macrophages RAW264.7 cells. The relative expression of PDCD4 mRNA was detected by qPCR after 50pM PDCD4 siRNA, including si-01, si-02, and si-03, and negative control (NC) was transfected into RAW264.7 cells for 48 h, respectively: ∗ p < 0.05 versus control, ∧ p < 0.05 versus NC group. (b) Western blot analyzed the protein level of PDCD4 when RAW264.7 cells had been treated by 50pM PDCD4 siRNA (si-02) and NC for 48 h. Representative bands show the protein expression of PDCD4 (the upper bands), β-actin (the lower bands) in left panel, and the histograms in right panel showing quantification of PDCD4 bands normalization relative to β-actin expression in (b): ∗ p < 0.05 versus NC group. (c, d, and e) qPCR analyzed the mRNA expression of TNF-α, IL-6, and IL-10 in RAW264.7 cells. They were transfected into the anti-miR-155 or PDCD4 siRNA for 24 h and then treated by ox-LDL for 24 h: ∗ p < 0.05 versus control, ∧ p < 0.05 versus 20 μg/mL ox-LDL group, and # p < 0.05 versus 20 μg/mL ox-LDL plus PDCD4 siRNA group. The data are presented as the mean ± SE of three separate experiments.
Figure 4 Downregulation of PDCD4 and miR-155 regulated the expression of IL-6, TNF-α, and IL-10. (a) Knocking down the expression of PDCD4 mRNA by PDCD4 siRNA in macrophages RAW264.7 cells. The relative expression of PDCD4 mRNA was detected by qPCR after 50pM PDCD4 siRNA, including si-01, si-02, and si-03, and negative control (NC) was transfected into RAW264.7 cells for 48 h, respectively: ∗ p < 0.05 versus control, ∧ p < 0.05 versus NC group. (b) Western blot analyzed the protein level of PDCD4 when RAW264.7 cells had been treated by 50pM PDCD4 siRNA (si-02) and NC for 48 h. Representative bands show the protein expression of PDCD4 (the upper bands), β-actin (the lower bands) in left panel, and the histograms in right panel showing quantification of PDCD4 bands normalization relative to β-actin expression in (b): ∗ p < 0.05 versus NC group. (c, d, and e) qPCR analyzed the mRNA expression of TNF-α, IL-6, and IL-10 in RAW264.7 cells. They were transfected into the anti-miR-155 or PDCD4 siRNA for 24 h and then treated by ox-LDL for 24 h: ∗ p < 0.05 versus control, ∧ p < 0.05 versus 20 μg/mL ox-LDL group, and # p < 0.05 versus 20 μg/mL ox-LDL plus PDCD4 siRNA group. The data are presented as the mean ± SE of three separate experiments.CC BY · PubMed Central
Figure 5 miR-155 regulated directly SOCS1-STAT3 signal pathway. (a) Schematic of the interaction sites of 3′ UTR SOCS1 wild-type (WT) or mutation (Mut) with miR-155. (b) Luciferase reporter assay was used to validate miR-155 binding SOCS1 3′UTR in HEK293FT cells. The luciferase reporter plasmids carrying the WT or Mut 3′UTRof SOCS1 and miR-155 mimic/miR-negative control (NC) were cotransfected into HEK 293FT cells for 24 h, and then luciferase activity was detected: ∗ p < 0.05, relative to miR-NC group. (c) and (d) Western blot analyzed the protein expression of SOCS1 and p-STAT3 after miR-155 mimic, anti-miR-155, and miR-NC was transfected into RAW264.7 cells for 48 h, respectively: ∗ p < 0.05, relative to miR-NC group. The data are presented as the mean ± SE of three separate experiments.
Figure 5 miR-155 regulated directly SOCS1-STAT3 signal pathway. (a) Schematic of the interaction sites of 3′ UTR SOCS1 wild-type (WT) or mutation (Mut) with miR-155. (b) Luciferase reporter assay was used to validate miR-155 binding SOCS1 3′UTR in HEK293FT cells. The luciferase reporter plasmids carrying the WT or Mut 3′UTRof SOCS1 and miR-155 mimic/miR-negative control (NC) were cotransfected into HEK 293FT cells for 24 h, and then luciferase activity was detected: ∗ p < 0.05, relative to miR-NC group. (c) and (d) Western blot analyzed the protein expression of SOCS1 and p-STAT3 after miR-155 mimic, anti-miR-155, and miR-NC was transfected into RAW264.7 cells for 48 h, respectively: ∗ p < 0.05, relative to miR-NC group. The data are presented as the mean ± SE of three separate experiments.CC BY · PubMed Central
Figure 6 SOCS1-STAT3 pathway regulated PDCD4. (a) Fluorescence images (×100) showed RAW264.7 cells were infected with SOCS1 overexpression adenovirus (Adv. SOCS1) and vector adenovirus (Adv. GFP). (b) qPCR detected the SOCS1 mRNA expression after cells were infected with Adv. SOCS1 and Adv. GFP for 48 h: ∗ p < 0.05 versus control, ∧ p < 0.05 versus Adv. GFP group. (c) Downregulation of the expression of STAT3 mRNA by STAT3 siRNA in macrophages RAW264.7 cells. The relative expression of STAT3 mRNA was detected by qPCR after 50pM STAT3 siRNA, including si-01, si-02, and si-03, and negative control (NC) was transfected into RAW264.7 cells for 48 h, respectively: ∗ p < 0.05 versus control, ∧ p < 0.05 versus NC group. (d) Western blot analyzed the protein level of phospho-STAT3 (p-STAT3) when RAW264.7 cells had been treated by 50pM STAT3 siRNA (si-03) and NC for 48 h. Representative bands show the protein expression of STAT3 (the upper bands), β-actin (the lower bands) in left panel, and the histograms in right panel showing quantification of STAT3 bands' normalization relative to β-actin expression in (d). ∗ p < 0.05 versus NC group. (e) Western blot analyzed the protein level of SOCS1, p-STAT3, and PDCD4 in RAW264.7 cells. They were transfected into the Adv. SOCS1 or STAT3 siRNA for 24 h and then treated by ox-LDL for 24 h. Representative bands show the protein expression of SOCS1 (the upper bands), p-STAT3 (the middle bands), PDCD4 (the third bands), and β-actin (the lower bands) in left panel, and the histograms in right panel showing quantification of STAT3 bands' normalization relative to β-actin expression. ∗ p < 0.05 versus control, ∧ p < 0.05 versus 20 μg/mL ox-LDL group, and # p < 0.05 versus 20 μg/mL ox-LDL plus Adv. SOCS1 group. The data are presented as the mean ± SE of three separate experiments.
Figure 6 SOCS1-STAT3 pathway regulated PDCD4. (a) Fluorescence images (×100) showed RAW264.7 cells were infected with SOCS1 overexpression adenovirus (Adv. SOCS1) and vector adenovirus (Adv. GFP). (b) qPCR detected the SOCS1 mRNA expression after cells were infected with Adv. SOCS1 and Adv. GFP for 48 h: ∗ p < 0.05 versus control, ∧ p < 0.05 versus Adv. GFP group. (c) Downregulation of the expression of STAT3 mRNA by STAT3 siRNA in macrophages RAW264.7 cells. The relative expression of STAT3 mRNA was detected by qPCR after 50pM STAT3 siRNA, including si-01, si-02, and si-03, and negative control (NC) was transfected into RAW264.7 cells for 48 h, respectively: ∗ p < 0.05 versus control, ∧ p < 0.05 versus NC group. (d) Western blot analyzed the protein level of phospho-STAT3 (p-STAT3) when RAW264.7 cells had been treated by 50pM STAT3 siRNA (si-03) and NC for 48 h. Representative bands show the protein expression of STAT3 (the upper bands), β-actin (the lower bands) in left panel, and the histograms in right panel showing quantification of STAT3 bands' normalization relative to β-actin expression in (d). ∗ p < 0.05 versus NC group. (e) Western blot analyzed the protein level of SOCS1, p-STAT3, and PDCD4 in RAW264.7 cells. They were transfected into the Adv. SOCS1 or STAT3 siRNA for 24 h and then treated by ox-LDL for 24 h. Representative bands show the protein expression of SOCS1 (the upper bands), p-STAT3 (the middle bands), PDCD4 (the third bands), and β-actin (the lower bands) in left panel, and the histograms in right panel showing quantification of STAT3 bands' normalization relative to β-actin expression. ∗ p < 0.05 versus control, ∧ p < 0.05 versus 20 μg/mL ox-LDL group, and # p < 0.05 versus 20 μg/mL ox-LDL plus Adv. SOCS1 group. The data are presented as the mean ± SE of three separate experiments.CC BY · PubMed Central
Figure 7 Inhibition of miR-155 by Anti-miR-155 decreased the atherosclerotic development in ApoE−/− mice with HFD (ApoE−/− HFD mice). (a) qPCR detected the miR-155 and CD68 mRNA expression in aortic tissue when ApoE−/− HFD mice were injected with anti-miR-155 and anti-NC. ∗ p < 0.05 versus anti-NC. (b) Atherosclerotic plaques in the thoracoabdominal aorta of anti-miR-155 and anti-NC-injected ApoE−/− HFD mice were assessed by Oil Red O staining, and the plaques areas were quantified by using Imagepro-plus 7.0: ∗ p < 0.05 versus anti-NC. (c) Western blot analyzed the protein level of SOCS1, p-STAT3, and PDCD4 in aortic tissue when ApoE−/− HFD mice were injected with anti-miR-155 and anti-NC for 2 weeks. Representative bands show the protein expression of SOCS1 (the upper bands), p-STAT3 (the middle bands), PDCD4 (the third bands), and β-actin (the lower bands) in the left panel, and the histograms in the right panel show quantification of the bands' normalization relative to β-actin expression in (c). The data are presented as the mean ± SE of three separate experiments: ∗ p < 0.05 versus anti-NC. (d) The plasma level of IL-6, TNF-α, and IL-10 of anti-miR-155 and anti-NC-injected ApoE−/− HFD was detected by the ELISA kit (n = 4): ∗ p < 0.05 versus ApoE−/− HFD group, ∧ p < 0.05 versus anti-NC-injected ApoE−/− HFD group. (e) qPCR analyzed the expression of IL-6, TNF-α, and IL-10 of c57 mice, ApoE−/− ND, ApoE−/− HFD, anti-miR-155, and anti-NC-injected ApoE−/− HFD mice in aortic tissue (n = 4): ∗ p < 0.05, relative to c57 mice WT, ∧ p < 0.05, relative to ApoE−/− ND, # p < 0.05, relative to ApoE−/− HFD.
Figure 7 Inhibition of miR-155 by Anti-miR-155 decreased the atherosclerotic development in ApoE−/− mice with HFD (ApoE−/− HFD mice). (a) qPCR detected the miR-155 and CD68 mRNA expression in aortic tissue when ApoE−/− HFD mice were injected with anti-miR-155 and anti-NC. ∗ p < 0.05 versus anti-NC. (b) Atherosclerotic plaques in the thoracoabdominal aorta of anti-miR-155 and anti-NC-injected ApoE−/− HFD mice were assessed by Oil Red O staining, and the plaques areas were quantified by using Imagepro-plus 7.0: ∗ p < 0.05 versus anti-NC. (c) Western blot analyzed the protein level of SOCS1, p-STAT3, and PDCD4 in aortic tissue when ApoE−/− HFD mice were injected with anti-miR-155 and anti-NC for 2 weeks. Representative bands show the protein expression of SOCS1 (the upper bands), p-STAT3 (the middle bands), PDCD4 (the third bands), and β-actin (the lower bands) in the left panel, and the histograms in the right panel show quantification of the bands' normalization relative to β-actin expression in (c). The data are presented as the mean ± SE of three separate experiments: ∗ p < 0.05 versus anti-NC. (d) The plasma level of IL-6, TNF-α, and IL-10 of anti-miR-155 and anti-NC-injected ApoE−/− HFD was detected by the ELISA kit (n = 4): ∗ p < 0.05 versus ApoE−/− HFD group, ∧ p < 0.05 versus anti-NC-injected ApoE−/− HFD group. (e) qPCR analyzed the expression of IL-6, TNF-α, and IL-10 of c57 mice, ApoE−/− ND, ApoE−/− HFD, anti-miR-155, and anti-NC-injected ApoE−/− HFD mice in aortic tissue (n = 4): ∗ p < 0.05, relative to c57 mice WT, ∧ p < 0.05, relative to ApoE−/− ND, # p < 0.05, relative to ApoE−/− HFD.CC BY · PubMed Central

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  1. 10.3892/or-00000838 · DOI
  2. 2011, Science Signaling, 4

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Metadata

Authors
Ye, J., Guo, R., Shi, Y., Qi, F., Guo, C., & Yang, L.
Affiliations
  • Army Medical University (CN)
  • Kunming General Hospital of Chengdu Military Command (CN)
  • Department of Cardiology, Kunming General Hospital of Chengdu Military Area, Yunnan 650032, China
Corresponding author
Yang, Lixia
ORCID
Venue
Mediators of Inflammation
Publisher
Wiley
Published
2016
Volume
2016
Pages
1-14
ISSN
  • 0962-9351 (ISSN-L)
  • 1466-1861
DOI
10.1155/2016/8060182
PMC
PMC5098093
OpenAlex
W2533066341
Cited by
127
References
49
Citations by year
2026: 11 · 2025: 11 · 2024: 14 · 2023: 19 · 2022: 11 · 2021: 9 · 2020: 21 · 2019: 18 · 2018: 8 · 2017: 5
FWCI
4.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Mediators of Inflammation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
Keywords
miR-155, atherosclerosis, atherogenesis, inflammation, SOCS1, STAT3, PDCD4, macrophages, interleukin-6, TNF-α, oxidized LDL, microRNA
MeSH terms
RAW 264.7 Cells, Suppressor of Cytokine Signaling 1 Protein, Animals, Aorta, Apolipoproteins E, Inflammation, Male, Tumor Necrosis Factor-alpha, Signal Transduction, RNA-Binding Proteins, RNA, Small Interfering, MicroRNAs, Atherosclerosis, STAT3 Transcription Factor, Apoptosis Regulatory Proteins, Mice

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