Chromosome 21-derived hsa-miR-155-5p regulates mitochondrial biogenesis by targeting Mitochondrial Transcription Factor A (TFAM)
Adolfo Quiñones-Lombraña and Javier G. Blanco*
Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York
Abstract
The regulation of mitochondrial biogenesis is under the control of nuclear genes including the
master mitochondrial transcription factor A (TFAM). Recent evidence suggests that the expression
of TFAM is regulated by microRNAs (miRNAs) in various cellular contexts. Here, we show that
hsa-miR-155-5p, a prominent miRNA encoded in chromosome 21, controls the expression of
TFAM at the post-transcriptional level. In human fibroblasts derived from a diploid donor,
downregulation of TFAM by hsa-miR-155-5p decreased mitochondrial DNA (mtDNA) content. In
contrast, downregulation of TFAM by hsa-miR-155-5p did not decrease mtDNA content in
fibroblasts derived from a donor with Down syndrome (DS, trisomy 21). In line, downregulation
of mitochondrial TFAM levels through hsa-miR-155-5p decreased mitochondrial mass in diploid
fibroblasts but not in trisomic cells. Due to the prevalence of mitochondrial dysfunction and
cardiac abnormalities in subjects with DS, we examined the presence of potential associations
between hsa-miR-155-5p and TFAM expression in heart samples from donors with and without
DS. There were significant negative associations between hsa-miR-155-5p and TFAM expression
in heart samples from donors with and without DS. These results suggest that regulation of TFAM
by hsa-miR-155-5p impacts mitochondrial biogenesis in the diploid setting but not in the DS
setting.
Keywords
TFAM; Hsa-miR-155-5p; mitochondrial biogenesis; Down syndrome
© 2015 Published by Elsevier B.V.*Corresponding author at: Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, 470 Kapoor Hall, Buffalo, NY 14214 – 8033, USA, Phone: 716 - 645 - 4820, Fax: 716 - 829 - 6569, [email protected].
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Conflicts of interestThe authors declare that there are no conflicts of interest.
HHS Public AccessAuthor manuscriptBiochim Biophys Acta. Author manuscript; available in PMC 2016 July 01.
Published in final edited form as:Biochim Biophys Acta. 2015 July ; 1852(7): 1420–1427. doi:10.1016/j.bbadis.2015.04.004.
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1. Introduction
TFAM (Mitochondrial Transcription Factor A) is a nucleus-encoded protein that plays a
pivotal role in the transcription and maintenance of mitochondrial DNA (mtDNA) [1, 2].
TFAM regulates mtDNA copy number and mitochondria biogenesis [3–5]. Reports have
shown that the expression of TFAM is in part regulated by specific microRNAs (miRNAs).
miRNAs are small noncoding RNAs that control the expression of target genes through
mechanisms involving the suppression of mRNA translation or the stimulation of mRNA
degradation [6]. For example, miR-23b down-regulates TFAM in glioma cells which in turn
leads to inhibition of cell proliferation, cell cycle progression, migration and colony
formation [7]. Yamamoto et al. have shown that miR-494 regulates mitochondrial biogenesis
in skeletal muscle through TFAM and Forkhead box j3 [8]. A recent study by Yao et al.
identified miR-200a as a regulator of TFAM expression and mtDNA copy number in breast
cancer cells [9].
Hsa-miR-155, a multifunctional microRNA encoded by chromosome 21, is involved in
various biological and pathological processes including immunity and inflammation [10,
11]. For example, the overexpression of hsa-miR-155 in damaged hearts after viral
myocarditis has been linked to adverse cardiac immune activation [12]. The chromosomal
location of hsa-miR-155 has motivated studies to elucidate its potential contribution to the
complex pathophysiology of Down syndrome (DS, trisomy 21) [13]. Recently, we
quantitated the expression of hsa-miR-155 in heart tissue and found no differences between
samples from donors with and without DS [14]. We postulated that inter-individual
variability together with factors that impact global gene expression in trisomy 21 (e.g.,
chromatin accessibility) may offset the expected gene-dosage increase for cardiac hsa-
miR-155 expression in the DS setting [15].
Mitochondrial dysfunction has been noted as a possible contributor to the DS phenotype
[16]. For example, Coskun et al. described decreased mtDNA in brain tissue from donors
with DS and dementia [17]. We have documented an average 33% decrease in cardiac
mtDNA content in samples from donors with DS in comparison to age-matched samples
from donors without DS; although the trend towards decreased cardiac mtDNA content did
not reach statistical significance (P > 0.05), we speculated that subtle decreases in cardiac
mtDNA may have pathophysiological relevance in the context of DS [14]. Additional lines
of evidence for mitochondrial dysfunction in DS include changes in the expression of genes
involved in the Krebs cycle and oxidative phosphorylation, and reductions in the activity of
mitochondrial enzymes [18–23]. Thus, the essential role of TFAM during maintenance of
mtDNA, the evidence in support of mitochondrial dysfunction in DS, and the potential
contribution of hsa-miR-155 to the DS phenotype lead us to hypothesize that TFAM may be
differentially regulated by hsa-miR-155 in the DS setting. Therefore, the goal of this study
was to test whether hsa-miR-155 regulates TFAM expression and mitochondrial biogenesis
in cell lines derived from subjects with and without DS. Complementary studies were
performed to test for associations between TFAM and hsa-miR-155 expression in heart
samples from donors with and without DS.
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2. Methods
2.1. Cell culture and reagents
CHO-K1 cells (Chinese hamster ovary-derived cell line, CCL-61) were obtained from the
American Type Culture Collection (Manassas, VA). Fibroblast cell lines derived from
donors with (GM01920) and without DS (GM00323) were obtained from the Coriell
Institute for Medical Research (Camden, NJ). Cell culture reagents were purchased from
Life Technologies (Carlsbad, CA). Fibroblast cell lines were routinely cultured in T75 flasks
using DMEM medium. CHO-K1 cells were grown in F12K medium. DMEM and F12K
media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-
Aldrich, St. Louis, MO), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cultures were
grown and maintained at low passage numbers (n < 12) using standard incubation conditions
at 37°C, 5% CO2, and 95% relative humidity.
2.2. Constructs and Site-Directed Mutagenesis
The microRNA mimic and hairpin inhibitor for hsa-miR-155-5p, as well as mimic and
inhibitor negative controls, were obtained from GE). The full-length TFAM 3′-UTR
construct was synthesized by OriGene (Rockville, MD). A 1078 bp TFAM 3′-UTR fragment
was cloned into a pMirTarget vector (OriGene) downstream of the firefly luciferase gene.
The QuikChange II XL-site-directed mutagenesis kit (Agilent, Santa Clara, CA) was used to
generate a construct containing the 3’-UTR of TFAM with a mutated seed sequence for
hsamiR-155-5p. The following primers were used for site-directed mutagenesis: forward
primer 5′-CCTTATATTATGGATCCAGGAGTTTCGTTTTC-3′, and reverse primer 5′-
GAAAACGAAACTCCTGGATCCATAATATAAGG-3′ (the mutated bases are
underlined). All constructs were verified by DNA sequencing.
2.3. Transfections
Twenty four hours prior to transfections, CHO-K1 cells were plated in 24-well plates.
CHOK1 cells were co-transfected with the TFAM 3′-UTR or the mutant TFAM 3’-UTR
(mutTFAM 3’-UTR) luciferase reporter construct (50 ng) plus the internal control plasmid
pRL-TK (5 ng) and hsa-miR-155-5p miRNA mimic (5 nM), in the presence or absence of
the specific hsa-miR-155-5p inhibitor (50 nM), using DharmaFECT Duo transfection
reagent (GE). Identical concentrations of miRNA mimic and inhibitor negative controls
were used. Twenty four hours post-transfection, cultures were washed once with phosphate-
buffered saline (PBS) solution; cells were lysed in freshly diluted passive lysis buffer (100
µl/well. Promega, Madison, WI) by incubating the plates at room temperature on a shaker at
200 rpm for 60 minutes. Luciferase reporter gene activities were determined with the Dual-
Luciferase Reporter Assay System (Promega) per the manufacturer's instructions. Light
intensity was measured in a Synergy HT luminometer equipped with proprietary software
for data analysis (BioTek, Winooski, VT). Corrected firefly luciferase activities were
normalized to renilla luciferase activities and expressed as fold increases with respect to the
values obtained from control transfections with miRNA mimic negative control or the
combination of miRNA mimic and inhibitor negative controls.
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Twenty four hours prior transfections, fibroblast cells (GM00323 and GM01920) were
plated at 50% confluence in 24-well plates. Cells were co-transfected with hsa-miR-155-5p
mimic or miRNA mimic negative control (5 nM) in the presence or absence of specific hsa-
miR-155-5p inhibitor (50 nM) or inhibitor negative control using DharmaFECT Duo
transfection reagent. Cells were re-transfected after 48 hours and collected 96 hours after the
initial transfection.
2.4. Isolation of cytosolic and mitochondrial protein
Cytosolic protein extraction was performed using the NE-PER Nuclear and Cytoplasmic
Extraction Reagents kit (Pierce Biotechnology, Rockford, IL) according to the
manufacturer’s instructions. Mitochondria were isolated from fibroblast cells by differential
centrifugation with an isolation kit for cultured cells (Abcam, Cambridge, MA, USA).
Briefly, samples were homogenized in isolation buffer on ice, and the homogenates were
centrifuged at 1000 g for 10 min at 4 °C. The supernatants were centrifuged at 12000 g for
15 min at 4 °C to obtain the mitochondrial pellet. Protein concentrations were determined
with the bicinchoninic acid protein assay (Pierce).
2.5. TFAM content in mitochondria
Mitochondrial TFAM content was measured with the enzyme-linked immunosorbent assay
TFAM SimpleStep (Abcam) per the manufacturer’s instructions. Measurements were done
in quadruplicates.
2.6. Mitochondrial citrate synthase activity
Mitochondrial citrate synthase activity was measured with an immunocapture based assay
(Abcam) by recording the color development of 5-thio-2-nitrobenzoic acid (TNB) at 412
nm. Measurements were done in triplicates.
2.7. Mitochondrial content in fibroblasts
Mitotracker RED CMXRos (Life Technologies) was added to cell culture medium at a final
concentration of 20 nM. Cells were incubated for 30 min and fixed in 4% paraformaldehyde
for 15 min. Fixed cells were washed 3 times with PBS (0.1 M) and mounted onto glass
slides using Fluorsave (Calbiochem, San Diego, CA). Cell images were captured with a
Zeiss Axiovert 200 fluorescence microscope equipped with an Axiocam MRC camera (Carl
Zeiss, Jena, Germany). Cellular area and mitochondrial content were measured with the
ImageJ image analysis software v1.43 (NIH, Bethesda, MD, http://rsb.info.nih.gov/ij/) and
associated plug-ins as reported [24].
2.8. Cell viability
The viability of transfected fibroblast cells was assessed by recording the reduction of 3-
(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT. Sigma). Briefly, cells
were plated in 96 well plates and transfected as previously described. Then, 20 µL of MTT
solution (5 mg/mL) were added to each well followed by incubation at 37°C for 4 h. After
incubation, the medium was removed and 100 µL of DMSO were added into each well. The
plate was gently rotated on an orbital shaker for 10 min to dissolve the MTT precipitate. The
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absorbance at 570 nm was recorded with a Synergy HT microplate reader (BioTek). Cellular
viability was expressed as percentages relative to control incubations.
2.9. Heart Samples
The Institutional Review Board of the State University of New York at Buffalo approved
this research. Heart samples from donors with (n = 11) and without DS (n = 32) were
procured from The National Disease Research Interchange (NDRI, funded by the National
Center for Research Resources), The Cooperative Human Tissue Network (CHTN, funded
by the National Cancer Institute), and the National Institute of Child Health and Human
Development (NICHD) Brain and Tissue Bank. The main demographics from donors with-
and without-DS are summarized in supplemental Table 1. The postmortem to tissue
recovery interval was ≤ 10 h. Samples (2 – 20 g, myocardium, left ventricle only) were
frozen immediately after recovery and stored in liquid nitrogen until further processing. DS
status (yes/no) was obtained from anonymous medical records and confirmed by
comparative array hybridizations as described [14, 25, 26]. High quality RNA was isolated
with an automatic QuickGene-810 purification system (Autogen/FujiFilm, Holliston, MA).
Purity and integrity of the RNA templates was assessed by measuring A260/A280 ratio and
by gel electrophoresis in denaturing conditions following MIQE guidelines [27].
2.10. Quantification of mtDNA content and hsa-miR-155-5p expression
mtDNA content and the expression of hsa-miR-155-5p in the collection of heart samples
from donors with and without DS have been recently reported by us [14]. mtDNA content in
cultured cells was measured with a qRT-PCR based on the amplification of the
mitochondrial gene MT-ND1 and the nuclear gene 18S rRNA as reported [14]. The
expression of hsa-miR-155-5p in cultured cells was measured by qRT-PCR with specific
primers as reported [14].
2.11. Quantification of TFAM mRNA expression
TFAM mRNA expression was analyzed by qRT-PCR with specific primers (TFAMforward:
5’-GCGCTCCCCCTTCAGTTTTG-3'; TFAMreverse: 5’-
GTTTTTGCATCTGGGTTCTGAGC-3'). Briefly, total RNA (2.5 ng) was reverse
transcribed and amplified with one-step QuantiTect SYBR Green RT-PCR kits (Qiagen,
Venlo, The Netherlands). TFAM and ACTB (reference gene, ACTBforward: 5’-
GGACTTCGAGCAAGAGATGG-3', and ACTBreverse: 5'-
AGCACTGTGTTGGCGTACAG-3') were amplified in parallel in an iQ5 thermal cycler
(Bio-Rad) with the following cycling parameters: 50°C for 30 minutes (reverse
transcription), 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for
30 seconds and 72°C for 30 seconds. Calibration curves were prepared to analyze linearity
(r2 > 0.98) and PCR efficiency for the amplification of TFAM (efficiency: 101%) and ACTB
(efficiency: 97%). For each sample, the averaged Ct values for TFAM were normalized
against the averaged Ct values for ACTB using the dCt method [28]. The expression of
TFAM in individual heart samples was expressed relative to the averaged expression of
TFAM in the group of heart samples from donors without DS (n = 32), which was assigned
an arbitrary value of 1.0.
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2.12. Bioinformatics
The full-length sequence of human TFAM 3′-UTR (NCBI Reference Sequence:
NM_003201.1) was retrieved from Entrez (http://www.ncbi.nlm.nih.gov/Entrez/). The
RNAhybrid prediction tool was used to identify the potential binding site for hsa-miR-155 in
TFAM 3′-UTR [29].
2.13. Data analysis
Statistics were computed with Excel 2013 (Microsoft Office; Microsoft, Redmond, WA) and
GraphPad Prism version 4.03 (GraphPad Software Inc., La Jolla, CA). The Kolmogorov–
Smirnov test was used to analyze the normality of datasets. The Student’s t test was used to
compare group means. Spearman’s rank correlation coefficient was used for the correlation
analysis of non-normally distributed data. Data are expressed as the mean ± standard
deviation (SD). Differences between means were considered to be significant at P < 0.05.
3. Results
3.1. Hsa-miR-155-5p binds to TFAM
Computational screenings with the RNAhybrid prediction tool were performed to pinpoint
potential interactions between hsa-miR-155 and the 3′-UTR of TFAM. A candidate region in
TFAM (NM_003201) located between nucleotides 1477 and 1495 showed an energetically
favorable free energy value (ΔΔG) of −18.7 Kcal/mol for the biding of hsa-miR-155-5p (Fig.
1A). Next, we tested whether hsa-miR-155-5p interacts with TFAM by performing luciferase
assays in CHO-K1 cells co-transfected with a reporter plasmid containing the 3’-UTR of
TFAM or the mutant version (mutTFAM 3’-UTR) and hsa-miR-155-5p. The co-transfection
of hsa-miR-155-5p decreased the luciferase activity of the TFAM 3’-UTR reporter construct
in comparison to control co-transfections (P < 0.001, Fig. 1B). The luciferase activity of the
TFAM 3’-UTR construct was rescued by co-transfection with a specific hairpin inhibitor for
hsa-miR-155-5p (Fig. 1B). Moreover, hsa-miR-155-5p-mediated repression of luciferase
activity was abolished when a mutation was introduced in two adjacent nucleotides within
the seed region of hsa-miR-155-5p (Fig. 1C).
3.2. Effect of hsa-miR-155-5p on TFAM mRNA expression and mtDNA content in fibroblast cell lines from donors with and without Down syndrome
Basal TFAM mRNA expression was significantly lower in the DS cell line than in the non-
DS cell line under standard cell culture conditions (TFAM mRNADS: 0.65 ± 0.27 relative
fold vs. TFAM mRNANon-DS: 1.00 ± 0.26; P < 0.05). In the non-DS cell line, transfection of
the hsa-miR-155-5p mimic decreased the expression of TFAM mRNA by 28% (P < 0.01),
and reduced mtDNA content by 50% (P < 0.001), in comparison to controls (Fig. 2A). In the
DS cell line, the hsamiR-155-5p mimic reduced TFAM mRNA expression by 51% in
comparison to controls (P < 0.001). The hsa-miR-155-5p mimic did not reduce mtDNA
content in the DS cell line in comparison to controls (Fig. 2A).
In both cell lines, inhibition of hsa-miR-155-5p expression by the specific inhibitor restored
TFAM mRNA levels (Fig. 2B). In addition, mtDNA content remained unaffected after
cotransfections with the hsa-miR-155-5p specific inhibitor plus the hsa-miR-155-5p mimic
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(Fig. 2B). In all cases, there were no significant changes in cell viability (Supplemental
Table 2).
3.3. Effect of hsa-miR-155-5p on mitochondrial and cytosolic TFAM content
In non-DS fibroblast cells transfected with the hsa-miR-155-5p mimic, there was a slight
12% reduction (P < 0.05) in mitochondrial TFAM protein levels in comparison to controls
(Fig. 3A). No significant changes in mitochondrial TFAM levels were observed in DS
fibroblasts transfected with the hsa-miR-155-5p mimic (Fig. 3A). Co-transfection with the
hsa-miR-155-5p mimic plus the specific inhibitor resulted in no changes in mitochondrial
TFAM protein levels in DS and non-DS fibroblasts (Fig. 3B). There were no significant
changes in cytosolic TFAM levels in DS and non-DS fibroblasts transfected with the hsa-
miR-155-5p mimic (Supplemental Fig. 1).
3.4. Effect of hsa-miR-155-5p on mitochondrial content
Citrate synthase activity, an exclusive marker of the mitochondrial matrix, is proportional to
the number of viable mitochondria. In the non-DS cell line, the hsa-miR-155-5p mimic
significantly decreased citrate synthase activity by 38% (P < 0.001) in comparison to control
incubations (Fig. 4A). The effect of hsa-miR-155-5p mimic on citrate synthase activity was
abolished by co-transfections with the specific hsa-miR-155-5p inhibitor (Fig. 4B). In
contrast, the hsa-miR-155-5p mimic exerted not significant changes in mitochondrial citrate
synthase activity in the DS cell line (Fig. 4A).
Mitochondrial staining with Mitotracker RED and quantitative imaging analyses were
performed to determine whether the effects of hsa-miR-155-5p on mtDNA content and
citrate synthase activity were paralleled by a decrease in the number of mitochondria. In
non-DS fibroblasts, transfection of the hsa-miR-155-5p mimic caused a 26% decrease in the
number of mitochondria in comparison to controls (P < 0.001; Fig. 5A). The effect of hsa-
miR-155-5p mimic was abolished when cells were transfected with the specific inhibitor of
hsa-miR-155-5p (Fig. 5A). In DS cells, transfections of the hsa-miR-155-5p mimic did not
significantly change the number of mitochondria (Fig. 5B).
3.5. Hsa-miR-155 and TFAM mRNA levels in human hearts from donors with and without Down syndrome
Recently, we reported no differences in mtDNA content and hsa-miR-155 expression
between heart tissue samples from donors with and without Down syndrome [14]. These
observations were extended by documenting the relative expression of cardiac TFAM
mRNA. The relative expression of TFAM mRNA was similar in heart samples from donors
with and without DS (DSTFAMmRNA: 1.29 ± 0.71 relative fold vs. non-DSTFAM mRNA: 1.00
± 0.64 relative fold; Student’s t test, P = 0.215. Fig. 6A). Further correlation analyses
revealed a significant negative association between hsa-miR-155 and TFAM mRNA
expression in hearts from donors without DS (Spearman’s regression coefficient, rS =
−0.619, P < 0.001. Fig. 6B). Similarly, there was a significant negative association between
hsa-miR-155 and TFAM mRNA expression in heart tissue samples from donors with DS
(Spearman’s regression coefficient, rS = −0.846, P < 0.01. Fig. 6C). There were no
significant associations between cardiac mtDNA content and relative TFAM mRNA
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expression in samples from donors with (Spearman’s regression coefficient, rS = −0.080, P
= 0.816) and without DS (Spearman’s regression coefficient, rS = −0.179, P = 0.327).
4. Discussion
Mitochondrial dysfunction may contribute to the DS phenotype [16]. There is a paucity of
reports on the role of specific miRNAs during the regulation of TFAM expression and
mitochondrial biogenesis in the DS setting. The aim of this study was to examine whether
hsamiR-155, a prominent miRNA encoded by chromosome 21, regulates mitochondrial
biogenesis through interactions with TFAM in cells from donors with and without DS.
First, we identified a candidate binding site for hsa-miR-155-5p in the 3’-UTR of TFAM.
Loeb et al. showed that 40% of the binding interactions of hsa-miR-155 with the
transcriptome of activated CD4+ T cells occur at sites without perfect complementary
matches (i.e., noncanonical sites), similarly to the binding site in TFAM (Fig. 1A) [30].
Next, the bioinformatics prediction was experimentally verified through gene reporter
experiments in CHO-K1 cells cotransfected with the 3’-UTR of TFAM and hsa-miR-155-5p
(Fig. 1.B). The 27% decrease in luciferase activity exerted by the hsa-miR-155-5p mimic is
in line with previous observations describing the impact of various miRNAs on TFAM
regulation under comparable experimental conditions. For example: a) Yao et al.
documented a 20% decrease in luciferase activity for hsamiR-200, b) Jiang et al.
documented a 36% decrease in luciferase activity for hsa-miR-23b, and c) Yamamoto et al.
documented a 16% in luciferase activity for hsa-miR-494 [7–9]. The specificity of the
binding site for hsa-miR-155-5p was confirmed by mutating the sequence of the seed region
in the 3’-UTR of TFAM (Fig. 1C). We also found that transfection of hsa-miR-155-5p into
fibroblast cells derived from a donor without DS induced significant decreases in TFAM
mRNA expression and mtDNA content (Fig. 2). The transfection of hsa-miR-155-5p also
led to a significant decrease in TFAM mRNA levels in fibroblasts from a donor with DS;
however, mtDNA content remained essentially unchanged (Fig. 2). Under standard cell
culture conditions, the average mtDNA content in DS fibroblasts was 64% lower than the
mtDNA content in non-DS fibroblasts (P < 0.001. Fig. 2). It appears that in DS cells, the
downregulation of TFAM mRNA expression by hsa-miR-155-5p is not followed by
significant reductions in mtDNA content. It is possible that in the DS setting, further
reductions in mtDNA content beyond a critical threshold (i.e., MT-ND1/18S ratio: 0.08 ±
0.03) would not be tolerated without a compromise in cellular viability. This notion is
supported by the following findings: a) transfection of hsa-miR-155-5p induced a slight but
consistent reduction in mitochondrial TFAM protein levels in non-DS cells, but no effect
was apparent in the DS cells (Fig. 3), b) mitochondrial citrate synthase activity was reduced
by hsa-miR-155-5p in non-DS cells but not in non-DS cells (Fig. 4), and c) hsa-miR-155-5p
reduced the number of mitochondria in non-DS cells but the miRNA mimic exerted no
significant effect on the number of mitochondria in DS cells (Fig. 5). Together, these results
suggest that hsa-miR-155-5p is a negative regulator of TFAM expression and mitochondrial
biogenesis in diploid cells. As noted, there is an increasing number of reports pinpointing
specific miRNAs that regulate TFAM in various cellular contexts. Loeb et al. postulated that
combinations of miRNAs binding through canonical and noncanonical sites may afford a
wide spectrum of gene regulation with major biological consequences [30]. In line with this
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notion, it appears that the relatively modest regulation of TFAM expression by hsa-
miR-155-5p through a non-canonical interaction triggers important changes in mitochondrial
parameters (e.g., mtDNA and mitochondrial content). However, it is also possible that the
observed effects may result from additional interactions between hsa-miR-155-5p and
transcriptional factors involved in mitochondrial biogenesis. For example, PGC-1a is a
central inducer of mitochondrial biogenesis in cells, and the expression of PGC-1a is
reduced in brown fat cells that overexpress miR-155 [31]. Our results also suggest that hsa-
miR-155-5p interacts with TFAM in trisomic cells; however, the absence of significant
changes in specific mitochondrial parameters (e.g., mtDNA content, citrate synthase
activity, and number of mitochondria) points towards the involvement of additional factors
during the control of mitochondrial biogenesis in the DS setting.
Widespread mitochondrial dysfunction in DS has been associated with the development of
cardiac alterations in structure and function in some individuals with DS [32]. In this study,
we identified significant associations between cardiac hsa-miR-155 and TFAM mRNA
expression in heart tissue samples from donors with and without DS, with no significant
alterations in mtDNA content (Fig. 6). Although these pilot observations are limited by the
relatively small number of samples from donors with DS, the extent of the linear
associations in heart tissue provides additional support for a distinct functional link between
hsa-miR-155-5p and TFAM. Donor’s age is a relevant co-variable when analyzing changes
in mtDNA content in human tissues [22]. For example, we have reported a more pronounced
decline in cardiac mtDNA content with aging in samples from donors without DS than in
samples from donors with DS [14]. The current limitations in sample size precluded us from
examining the combined impact of donor’s age, hsa-miR-155-5p and TFAM expression on
cardiac mtDNA through multiple linear regression analysis.
Our findings suggest a novel function for hsa-miR-155-5p during the control of TFAM
expression and mitochondrial biogenesis in diploid cells. In contrast, it appears that the
hsamiR-155-5p-TFAM interaction does not impact the biogenesis of mitochondria in cells
with trisomy 21.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
Research in this report was supported by the National Institute of General Medical Sciences and the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under awards R01GM073646 and R03HD076055. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Highlights
• Hsa-miR-155-5p, a miRNA encoded in chromosome 21, controls the expression
of TFAM.
• Hsa-miR-155-5p and TFAM expression correlates in hearts with and without
trisomy 21.
• Hsa-miR-155-5p regulates TFAM and mitochondrial biogenesis in the diploid
setting.
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Fig. 1. Schematic representation of human TFAM showing the potential binding site for hsa-
miR-155-5p (A, top). Predicted sequence interactions between TFAM and hsa-miR-155-5p,
and between the mutated TFAM 3’-UTR (mutTFAM 3’-UTR) and hsa-miR-155-5p (A,
middle). Luciferase activities of the TFAM 3’-UTR construct in CHO-K1 cells transfected
with hsa-miR-155-5p mimic or specific controls (B). Luciferase activities of the mutTFAM
3’-UTR construct in CHO-K1 cells transfected with hsa-miR-155-5p mimic or specific
controls (C). For each construct, normalized luciferase activities were expressed relative to
the values from control transfections (miRNA mimic negative control or miRNA mimic
negative control + inhibitor negative control). Data represent the mean ± standard deviation
of three independent experiments. ***P < 0.001, (Student's t-test).
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Fig. 2. TFAM mRNA levels (left) and mtDNA content (right) in fibroblast cell lines derived from
donors with and without DS. Fibroblast cells were transfected with hsa-miR-155-5p mimic
(A) or hsa-miR-155-5p plus the specific hsa-miR-155-5p inhibitor control (B). Data
represent the mean ± SD from three independent experiments performed in triplicate. **P <
0.01, ***P < 0.001, (Student's t-test).
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Fig. 3. TFAM protein levels in mitochondria from fibroblast cell lines derived from donors with
and without DS. Fibroblast cells were transfected with hsa-miR-155-5p mimic (A) or hsa-
miR-155-5p plus the specific hsa-miR-155-5p inhibitor control (B). Data represent the mean
± SD of four independent experiments. *P < 0.05 (Student's t-test).
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Fig. 4. Mitochondrial citrate synthase activity in fibroblast cell lines derived from donors with and
without DS. Fibroblast cells were transfected with hsa-miR-155-5p mimic (A) or hsa-
miR-155-5p plus the specific hsa-miR-155-5p inhibitor control (B). Data represent the mean
± SD of four independent experiments. ***P < 0.001, (Student's t-test).
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Fig. 5. Mitochondrial content in fibroblast cell lines derived from donors without (A) and with DS
(B). Fibroblast cells were transfected with hsa-miR-155-5p mimic (top panels) or hsa-
miR-155-5p plus the specific hsa-miR-155-5p inhibitor control (bottom panels).
Representative fluorescence microscopy images are shown in the left and corresponding
graphs in the right. For each condition, the graphs show data from ten cells per experiment,
and from three independent experiments. Each symbol depicts the number of mitochondria
per square micrometer of a whole single cell. ***P < 0.001, (Student's t-test).
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Fig. 6. Cardiac TFAM mRNA expression in samples from donors with (n = 11) and without DS (n
= 32). Each symbol depicts the average of individual samples. Samples were analyzed in
triplicates (A). Linear regression analysis of cardiac hsa-miR-155 expression versus TFAM
mRNA expression in samples from donors without (B) and with DS (C).
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