Epigenetic alteration of Mir-122 and let-7b expression in Adipose Tissue-Derived Mesenchymal Stem Cells by
Trichostatin A
E. Alizadeh1, N. Zarghami1, M.B. Eslaminejad2, M. Abasi1, A. Barzegar3, S. Jahangir2, S. Hashemzadeh4
1) Department of Medical Biotechnology, School of Advanced Medical Science, Tabriz University of Medical Science, Tabriz, Iran 2) Department of Stem Cell and
Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 3) Research Institute for Fundamental Sciences (RIFS), University of
Tabriz, Tabriz, Iran4) Department of General Surgery, Faculty of Medicine, Tabriz University of Medical Science, Tabriz, Iran - zarghami@tbzmed.ac.ir
Results
Objectives
Methods
Subcutaneous adipose-tissue was obtained with
informed consent during surgery from 6 donors in
Teaching-Imam Reza Hospital of Tabriz, Iran(Table 1).
Isolation of AMSCs was performed using enzyme
digestion and established-protocol. AMSCs were
stained with combinations of antibodies conjugated
with FITC or PE: CD34, MHCII, CD44, CD11b, CD45,
and CD90 followed by flow cytometry analysis(Fig 2).
Differentiation potential of AMSCs was evaluated by
osteogenic and adipogenic induction.(Fig1 A,B)
AMSCs were cultured in media containing L-DMEM,
EGF, OMS, ITS, bFGF, and various concentrations (025µM) of TSA. The colony-forming and MTT assays
was performed. The expression of mir-122, let-7a, let7b, let-7c, and let-7d was investigated by LNA-based
Real time PCR in AMSCs, at days 7th, 14th and 21st
after TSA treatments.
Our isolated AMSCs expressed CD44, CD73, and CD90
markers (95-97%). Epithelial-like morphology was
observed in AMSCs, surrounded by fibroblastic-cells 20
days after culturing with TSA(Fig 1D). Additionally, among
the human miRNAs investigated by the real time PCR,
miR-122 was induced and conversely let-7b miRNAs
was down-regulated in the TSA-treated group as
compared to the control group.(Fig 3)
1
Age Gender
43 M
Tissue
Subcutaneous adipose
2
47
M
Subcutaneous adipose
3
53
F
Subcutaneous adipose
Figure -2. Analysis of surface markers by flow cytometry
3
2.5
4
45
F
Subcutaneous adipose
5
55
F
Subcutaneous adipose
6
58
F
Subcutaneous adipose
Tabel-1 Characteristics of donors
Expression(fold)
Adipose tissue derived-mesenchymal stem cells (AMSCs)
have the potential of differentiation into different lineages.
Abundance and non-invasive isolation of AMSCs made
them favorite choice for autologus stem cell therapy.
MicroRNAs are small, non-coding RNAs with great impact
on proliferation and differentiation. Recent study pointed
that let-7-family are main microRNAs in exosomes deriving
from AMSCs. Also, miR-122 is liver-specific microRNA.
Histone-deacetylase inhibitors such as Trichostatin A are
epigenetic agents with differentiation-inducing properties.
The aim of this study was to investigate the effect of
Trichostatin A (TSA) on expression levels of mir-122, let7a, let-7b, let-7c and let-7d in AMSCs.
2
1.5
miR-122
let-7b
1
0.5
0
zero
7th
14th
21th
Days
Figure-3. Expression levels of miR-122 and Let7b by Real time PCR
References
Conclusions
Figure -1. Adipogenic(A), osteogenic(B) differentiation of
AMSCs(C), AMSCs after TSA treatments(D)
Our results imply that mir-122 and let-7b might have a
possible role in differentiation process. So, these
findings may be applicable in production of functional
hepatocytes from AMSCs utilizing microRNAs and
epigenetic agents.