transcription factor a ( hhs public access tfam biochim ...the regulation of mitochondrial...

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.

Quiñones-Lombraña and Blanco

Biochim Biophys Acta. Author manuscript; available in PMC 2016 July 01.

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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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transcription factor a ( hhs public access tfam biochim ...the regulation of mitochondrial...

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