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AGE

, 36:9724 | Cite as

miR-543 and miR-590-3p regulate human mesenchymal stem cell aging via direct targeting of AIMP3/p18

  • Seunghee Lee
  • Kyung-Rok Yu
  • Young-Sil Ryu
  • Young Sun Oh
  • In-Sun Hong
  • Hyung-Sik Kim
  • Jin Young Lee
  • Sunghoon Kim
  • Kwang-Won SeoEmail author
  • Kyung-Sun KangEmail author
Article

Abstract

Previously, AIMP3 (aminoacyl-tRNAsynthetase-interacting multifunctional protein-3) was shown to be involved in the macromolecular tRNA synthetase complex or to act as a tumor suppressor. In this study, we report a novel role of AIMP3/p18 in the cellular aging of human mesenchymal stem cells (hMSCs). We found that AIMP3/p18 expression significantly increased in senescent hMSCs and in aged mouse bone marrow-derived MSCs (mBM-MSCs). AIMP3/p18 overexpression is sufficient to induce the cellular senescence phenotypes with compromised clonogenicity and adipogenic differentiation potential. To identify the upstream regulators of AIMP3/p18 during senescence, we screened for potential epigenetic regulators and for miRNAs. We found that the levels of miR-543 and miR-590-3p significantly decreased under senescence-inducing conditions, whereas the AIMP3/p18 protein levels increased. We demonstrate for the first time that miR-543 and miR-590-3p are able to decrease AIMP3/p18 expression levels through direct binding to the AIMP/p18 transcripts, which further compromised the induction of the senescence phenotype. Taken together, our data demonstrate that AIMP3/p18 regulates cellular aging in hMSCs possibly through miR-543 and miR-590-3p.

Keywords

AIMP3/p18 Stem cell Aging miR-543 miR-590-3p 

Abbreviations

AIMP3

aminoacyl-tRNAsynthetase-interacting multifunctional protein-3

MSCs

mesenchymal stem cells

hUCB

human umbilical cord blood

mBM

mouse bone marrow

miRNAs

microRNAs

MRS

methionyl-tRNAsynthetase

SA-β-gal

senescence-associated β-galactosidase

ICC

immunocytochemistry

WT

wild type

TG

transgenic

VPA

valproic acid

SB

sodium butyrate

5-azaC

5-azacytidine

AceH4

acetyl histone H4

H3K4Me3

histone H3 lysine 4 trimethylation

H3K27Me3

histone H3 lysine 27 trimethylation

Notes

Acknowledgments

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: A120176) and by the Research Institute for Veterinary Science, Seoul National University.

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

11357_2014_9724_Fig1_ESM.jpg (91 kb)
Fig. S1

AIMP3/p18 expression in oxidative stress-induced senescent cells and proliferation rates in AIMP3/p18-overexpressing hMSCs. (A) hMSCs were treated with 400 μM H2O2 for 3 days. After treatment, SA-β-gal staining was performed. (B) After treatment with 400 μM H2O2 for the indicated periods, the expression levels of the AIMP3/p18, p16INK4A and p21CIP1/WAF1 proteins were quantified by western blot analysis. (C) After AIMP3/p18 overexpression, cell cycle analysis was performed (JPEG 90 kb)

11357_2014_9724_Fig2_ESM.jpg (199 kb)
Fig. S2

Changes in CDK inhibitors by AIMP3/p18 up- or down-regulation. (A) Primary cultures of mBM-MSCs from young (4-week-old) and old (19-month-old) mice were cultured as described in the Materials and Methods. Protein levels of AIMP3/p18, p16INK4A and p21CIP1/WAF1 were evaluated by western blot analysis. (B) Western blot analysis was performed on heart, liver and intestine tissues from 10-month-old WT and Aimp3/p18TG mice. (C) hMSCs were contact-inhibited for 4 days. Then, the cells were transfected with 50 nM siRNA-AIMP3/p18. A second transfection was performed without subculture after 2 days. The expression levels of the AIMP3/p18, p16INK4A and p21WAF1/CIP1 proteins were quantified by western blot analysis. (D) After transfection of the AIMP3/p18 expression vector, Lamin A/C and p16INK4A expression was investigated by western blot in Tera-1 cells. (E) After transfection of the AIMP3/p18 expression vector, p53 and p16INK4A expression was investigated by western blot in PC3 cells (JPEG 198 kb)

11357_2014_9724_Fig3_ESM.jpg (170 kb)
Fig. S3

Screening for the candidates for AIMP3/p18 regulation. (A) hMSCs were treated for 0, 1, 3 or 6 days with the following epigenetic regulators: 4 mM valproic acid (VPA), 2 mM sodium butyrate (SB) and 2 μM 5-aza-20-deoxycytidine (5-azaC). (B) The RNA expression levels of AIMP3/p18 in replicative- and mitomycin C-induced senescent cells were determined by real-time PCR. (C) Schematic diagram of the AIMP3/p18 promoter region. (D) Chromatin immunoprecipitation assays for acetyl histone H4 (AceH4), histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) in the AIMP3/p18 promoter region (C) in replicative- and mitomycin C-induced senescent cells. (*P <0.05; **P <0.01) (JPEG 169 kb)

11357_2014_9724_Fig4_ESM.jpg (390 kb)
Fig. S4

miR-543 and miR-590-3p are involved in AIMP3/p18 expression under senescence-inducing conditions. (A) The counterparts of the AIMP3/p18 mRNA 3′ UTR against seed regions of miRNAs. The numbers (left side) indicate matching locations. (B) miRNA sequences in various mammalian species. Red letters indicate binding sites to AIMP3/p18 mRNA. (C) The change in AIMP3/p18 expression relative to the passage number was quantified by western blot analysis. (D) The changes in miR-543 and miR-590-3p expression related to the passage number were quantified by real-time quantitative PCR. (E) AIMP3/p18 expression after miRNA transfection in contact-inhibited hMSCs. (F) mRNA level of AIMP3/p18 in mature and antisense miRNA-transfected hMSCs. (*P <0.05; **P <0.01) (JPEG 389 kb)

11357_2014_9724_Fig5_ESM.jpg (151 kb)
Fig. S4

miR-543 and miR-590-3p are involved in AIMP3/p18 expression under senescence-inducing conditions. (A) The counterparts of the AIMP3/p18 mRNA 3′ UTR against seed regions of miRNAs. The numbers (left side) indicate matching locations. (B) miRNA sequences in various mammalian species. Red letters indicate binding sites to AIMP3/p18 mRNA. (C) The change in AIMP3/p18 expression relative to the passage number was quantified by western blot analysis. (D) The changes in miR-543 and miR-590-3p expression related to the passage number were quantified by real-time quantitative PCR. (E) AIMP3/p18 expression after miRNA transfection in contact-inhibited hMSCs. (F) mRNA level of AIMP3/p18 in mature and antisense miRNA-transfected hMSCs. (*P <0.05; **P <0.01) (JPEG 389 kb)

11357_2014_9724_MOESM6_ESM.doc (59 kb)
Table S1 Primers used for real-time quantitative PCR analysis (DOC 59 kb)

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Copyright information

© American Aging Association 2014

Authors and Affiliations

  • Seunghee Lee
    • 1
    • 2
    • 3
  • Kyung-Rok Yu
    • 1
    • 2
  • Young-Sil Ryu
    • 1
    • 2
  • Young Sun Oh
    • 4
    • 5
  • In-Sun Hong
    • 6
    • 7
  • Hyung-Sik Kim
    • 1
    • 2
    • 3
  • Jin Young Lee
    • 1
    • 2
  • Sunghoon Kim
    • 4
    • 5
  • Kwang-Won Seo
    • 1
    • 2
    • 3
    Email author
  • Kyung-Sun Kang
    • 1
    • 2
    Email author
  1. 1.Adult Stem Cell Research Center, College of Veterinary MedicineSeoul National UniversitySeoulRepublic of Korea
  2. 2.Research Institute for Veterinary Medicine, College of Veterinary MedicineSeoul National UniversitySeoulRepublic of Korea
  3. 3.Institute for Stem Cell and Regenerative Medicine in Kang Stem Biotech, Biotechnology Incubating CenterSeoul National UniversitySeoulRepublic of Korea
  4. 4.Medicinal Bioconvergence Research CenterSeoul National UniversitySeoulRepublic of Korea
  5. 5.WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and TechnologySeoul National UniversitySuwonRepublic of Korea
  6. 6.Department of Molecular MedicineGachon UniversityIncheonRepublic of Korea
  7. 7.Lee Gil Ya Cancer and Diabetes InstituteIncheonRepublic of Korea

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