Nutr Res Pract.  2016 Aug;10(4):377-384. 10.4162/nrp.2016.10.4.377.

Involvement of miR-Let7A in inflammatory response and cell survival/apoptosis regulated by resveratrol in THP-1 macrophage

Affiliations
  • 1Department of Biomedical Engineering, Dongguk University, Gyeonggi 10326, Korea.
  • 2Department of Biology, York University, Toronto, Ontario, Canada.
  • 3Department of Food Science and Nutrition, Dong-A University, 37 Nakdong-daero 550beon-gil, Saha-gu, Busan 49315, Korea. oykim@dau.ac.kr

Abstract

BACKGROUND/OBJECTIVES
Resveratrol, a natural polyphenol, has multiple functions in cellular responses including apoptosis, survival, and differentiation. It also participates in the regulation of inflammatory response and oxidative stress. MicroRNA-Let-7A (miR-Let7A), known as a tumor suppressor miRNA, was recently reported to play a crucial role in both inflammation and apoptosis. Therefore, we examined involvement of miR-Let7A in the modulation of inflammation and cell survival/apoptosis regulated by resveratrol.
MATERIALS/METHODS
mRNA expression of pro-/anti-inflammatory cytokines and sirtuin 1 (SIRT1), and protein expression of apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, and caspase-3 and cleaved caspase-3 were measured, and cell viability and Hoechst/PI staining for apoptosis were observed in Lipopolysaccharide (LPS)-stimulated human THP-1 macrophages with the treatment of resveratrol and/or miR-Let7A overexpression.
RESULTS
Pre-treatment with resveratrol (25-200 µM) resulted in significant recovery of the reduced cell viabilities under LPS-induced inflammatory condition and in markedly increased expression of miR-Let7A in non-stimulated or LPS-stimulated cells. Increased mRNA levels of tumor necrosis factor-α and interleukin (IL)-6 induced by LPS were significantly attenuated, and decreased levels of IL-10 and brain-derived neurotrophic factor were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. Decreased expression of IL-4 mRNA by LPS stimulation was also significantly increased by miR-Let7A overexpression co-treated with resveratrol. In addition, decreased SIRT1 mRNA levels, and increased p-ASK1 levels and PI-positive cells by LPS stimulation were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination.
CONCLUSIONS
miR-Let7A may be involved in the inflammatory response and cell survival/apoptosis modulated by resveratrol in human THP-1 macrophages.

Keyword

Resveratrol; microRNA-Let7A; inflammation; ASK1; sirtuin 1

MeSH Terms

Apoptosis
Brain-Derived Neurotrophic Factor
Caspase 3
Cell Survival
Cytokines
Humans
Inflammation
Interleukin-10
Interleukin-4
Interleukins
Macrophages*
MAP Kinase Kinase Kinase 5
MicroRNAs
Necrosis
Oxidative Stress
RNA, Messenger
Sirtuin 1
Brain-Derived Neurotrophic Factor
Caspase 3
Cytokines
Interleukin-10
Interleukin-4
Interleukins
MAP Kinase Kinase Kinase 5
MicroRNAs
RNA, Messenger
Sirtuin 1

Figure

  • Fig. 1 Effect of resveratrol on cell viability of THP-1 macrophages under LPS induced inflammatory condition. The cell viability of THP-1 in all groups was measured using the MTT assay. The cell viability in LPS (1 µg/ml) stimulation was approximately 60%, whereas the resveratrol treatment groups showed higher cell viability (over 120%) compared to the normal group. Under LPS stimulated inflammatory condition, the cell viability of resveratrol-pretreated THP-1 cells (in all concentrations) was increased by over 30 % compared with that of only LPS treated THP-1 cells. Data are expressed as mean ± SEM and each experiment included 6 repeats per condition. * P < 0.05, ** P < 0.001 compared with normal control; φ P < 0.05 compared with LPS only stimulated cells

  • Fig. 2 Effect of resveratrol on miR-Let-7A expression in THP-1 macrophages under LPS induced inflammatory condition. Expression of miR-Let7A was measured in all groups using Tagman real time-PCR. Data are expressed as mean ± SEM and each experiment included 3 repeats per condition. * P < 0.05, ** P < 0.001 compared with normal control; φP < 0.05 compared with LPS only stimulated cells

  • Fig. 3 Resveratrol modulates mRNA expression of pro-inflammatory cytokines in cooperation with miR-Let7A. The mRNA levels of TNF-α and IL-6 in THP-1 cells were measured using quantitative real time PCR. Data are expressed as mean ± SEM and each experiment included 3 repeats per condition. * P < 0.05, ** P < 0.001 compared with normal control; φ P < 0.05 compared with LPS only stimulated cells; φφ P < 0.05 compared with LPS stimulated and miR Let7A overexpressed cells; Normal: non-treated control cells, Let7A (miR-Let7A mimic): Let7A overexpression, Resv: resveratrol (25 µM), LPS: lipopolysaccharide (1 µg/ml)

  • Fig. 4 Resveratrol modulates mRNA expression of BDNF and anti-inflammatory cytokines in cooperation with miR-Let7A. The mRNA levels of BDNF, IL-4, and IL-10 in THP-1 cells were measured using reverse transcription PCR. Data are expressed as mean ± SEM and each experiment included 3 repeats per condition. * P < 0.05, ** P < 0.001 compared with normal control; φ P < 0.05 compared with LPS only stimulated cells; φφ P < 0.05 compared with LPS stimulated and miR Let7A overexpressed cells; Normal: non-treated control cells, Let7A (miR-Let7A mimic): Let7A overexpression, Resv: resveratrol (25 µM), LPS: lipopolysaccharide (1 µg/ml)

  • Fig. 5 Resveratrol attenuates the activation of ASK1 in cooperation with miR-Let7A. Western blot analysis was performed for identification of apoptotic signaling in macrophages by resveratrol co-treated with or without miR-Let7A. Data were expressed as the mean ± SEM. and each experiment included 3 repeats per condition. φ P < 0.05 compared with LPS only stimulated cells.; Normal: non-treated cells, Let7A (miR-Let7A mimic): Let7A overexpression, Resv: resveratrol (25 µM), LPS: lipopolysaccharide (1 µg/ml)

  • Fig. 6 Inhibitory effect of resveratrol in cooperation with miR-Let7A on the activation of ASK1 in THP-1macrophages. Immunofluorescence staining was performed to check the activation of ASK1 (p-ASK1). Confocal microscopy analysis was performed to visualize the activation of ASK1 (p-ASK1). THP-1 cells were pretreated with resveratrol or/and Let7A mimic separately or following stimulation with LPS. p-ASK1 is represented by green staining, nuclear DNA is indicated by DAPI staining (blue color), and the combined images are presented; Normal: non-treated cells, Let7A (miR-Let7A mimic): Let7A overexpression, Resv: resveratrol (25 µM), LPS: lipopolysaccharide (1 µg/ml)

  • Fig. 7 Resveratrol stimulates SIRT1 pathways in cooperation with miR-Let7A. The mRNA levels of SIRT1 in THP-1 cells were measured using reverse transcription PCR. Data are expressed as mean ± SEM and each experiment included 3 repeats per conditions. * P < 0.05, ** P < 0.001 compared with normal control; φ P < 0.05 compared with LPS only stimulated cells; φφ P < 0.05 compared with LPS stimulated and miR Let7A overexpressed cells; Normal: non-treated cells, Let7A (miR-Let7A mimic): Let7A overexpression, Resv: resveratrol (25 µM), LPS: lipopolysaccharide (1 µg/ml)


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