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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Apr 11;1852(7):1420–1427. doi: 10.1016/j.bbadis.2015.04.004

Chromosome 21-derived hsa-miR-155-5p regulates mitochondrial biogenesis by targeting Mitochondrial Transcription Factor A (TFAM)

Adolfo Quiñones-Lombraña 1, Javier G Blanco 1,*
PMCID: PMC4433801  NIHMSID: NIHMS680325  PMID: 25869329

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

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.

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.

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 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.

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).

Fig. 1.

Fig. 1

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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).

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).

Fig. 2.

Fig. 2

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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).

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 (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).

Fig. 3.

Fig. 3

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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).

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).

Fig. 4.

Fig. 4

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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).

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).

Fig. 5.

Fig. 5

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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).

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 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).

Fig. 6.

Fig. 6

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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).

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 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

Supplemental data

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.

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.

Footnotes

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Conflicts of interest

The authors declare that there are no conflicts of interest.

References

  • 1.Falkenberg M, Larsson NG, Gustafsson CM. DNA replication and transcription in mammalian mitochondria. Annual review of biochemistry. 2007;76:679–699. doi: 10.1146/annurev.biochem.76.060305.152028. [DOI] [PubMed] [Google Scholar]
  • 2.Gensler S, Weber K, Schmitt WE, Perez-Martos A, Enriquez JA, Montoya J, Wiesner RJ. Mechanism of mammalian mitochondrial DNA replication: import of mitochondrial transcription factor A into isolated mitochondria stimulates 7S DNA synthesis. Nucleic acids research. 2001;29:3657–3663. doi: 10.1093/nar/29.17.3657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Matsushima Y, Goto Y, Kaguni LS. Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM) Proceedings of the National Academy of Sciences of the United States of America. 2010;107:18410–18415. doi: 10.1073/pnas.1008924107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Pohjoismaki JL, Wanrooij S, Hyvarinen AK, Goffart S, Holt IJ, Spelbrink JN, Jacobs HT. Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells. Nucleic acids research. 2006;34:5815–5828. doi: 10.1093/nar/gkl703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ekstrand MI, Falkenberg M, Rantanen A, Park CB, Gaspari M, Hultenby K, Rustin P, Gustafsson CM, Larsson NG. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Human molecular genetics. 2004;13:935–944. doi: 10.1093/hmg/ddh109. [DOI] [PubMed] [Google Scholar]
  • 6.Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nature reviews. Genetics. 2008;9:102–114. doi: 10.1038/nrg2290. [DOI] [PubMed] [Google Scholar]
  • 7.Jiang JD, Yang JF, Wang ZF, Wu GY, Liu F. TFAM is directly regulated by miR-23b in glioma. Oncol Rep. 2013;30:2105–2110. doi: 10.3892/or.2013.2712. [DOI] [PubMed] [Google Scholar]
  • 8.Yamamoto H, Morino K, Nishio Y, Ugi S, Yoshizaki T, Kashiwagi A, Maegawa H. MicroRNA-494 regulates mitochondrial biogenesis in skeletal muscle through mitochondrial transcription factor A and Forkhead box j3. Am J Physiol-Endoc M. 2012;303:E1419–E1427. doi: 10.1152/ajpendo.00097.2012. [DOI] [PubMed] [Google Scholar]
  • 9.Yao J, Zhou E, Wang Y, Xu F, Zhang D, Zhong D. microRNA-200a inhibits cell proliferation by targeting mitochondrial transcription factor A in breast cancer. DNA and cell biology. 2014;33:291–300. doi: 10.1089/dna.2013.2132. [DOI] [PubMed] [Google Scholar]
  • 10.Faraoni I, Antonetti FR, Cardone J, Bonmassar E. miR-155 gene: A typical multifunctional microRNA. Bba-Mol Basis Dis. 2009;1792:497–505. doi: 10.1016/j.bbadis.2009.02.013. [DOI] [PubMed] [Google Scholar]
  • 11.O'Connell RM, Taganov KD, Boldin MP, Cheng GH, Baltimore D. MicroRNA-155 is induced during the macrophage inflammatory response. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:1604–1609. doi: 10.1073/pnas.0610731104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Corsten MF, Papageorgiou A, Verhesen W, Carai P, Lindow M, Obad S, Summer G, Coort SLM, Hazebroek M, van Leeuwen R, Gijbels MJJ, Wijnands E, Biessen EAL, De Winther MPJ, Stassen FRM, Carmeliet P, Kauppinen S, Schroen B, Heymans S. MicroRNA Profiling Identifies MicroRNA-155 as an Adverse Mediator of Cardiac Injury and Dysfunction During Acute Viral Myocarditis. Circ Res. 2012;111:415-U155. doi: 10.1161/CIRCRESAHA.112.267443. [DOI] [PubMed] [Google Scholar]
  • 13.Sethupathy P, Borel C, Gagnebin M, Grant GR, Deutsch S, Elton TS, Hatzigeorgiou AG, Antonarakis SE. Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes. American journal of human genetics. 2007;81:405–413. doi: 10.1086/519979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hefti E, Quinones-Lombrana A, Redzematovic A, Hui J, Blanco JG. Analysis of mtDNA, miR-155 and BACH1 expression in hearts from donors with and without Down syndrome. Mitochondrial DNA. 2014:1–8. doi: 10.3109/19401736.2014.926477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Letourneau A, Santoni FA, Bonilla X, Sailani MR, Gonzalez D, Kind J, Chevalier C, Thurman R, Sandstrom RS, Hibaoui Y, Garieri M, Popadin K, Falconnet E, Gagnebin M, Gehrig C, Vannier A, Guipponi M, Farinelli L, Robyr D, Migliavacca E, Borel C, Deutsch S, Feki A, Stamatoyannopoulos JA, Herault Y, van Steensel B, Guigo R, Antonarakis SE. Domains of genome-wide gene expression dysregulation in Down's syndrome. Nature. 2014;508:345–350. doi: 10.1038/nature13200. [DOI] [PubMed] [Google Scholar]
  • 16.Valenti D, de Bari L, De Filippis B, Henrion-Caude A, Vacca RA. Mitochondrial dysfunction as a central actor in intellectual disability-related diseases: An overview of Down syndrome, autism, Fragile X and Rett syndrome. Neuroscience and biobehavioral reviews. 2014 doi: 10.1016/j.neubiorev.2014.01.012. [DOI] [PubMed] [Google Scholar]
  • 17.Coskun PE, Wyrembak J, Derbereva O, Melkonian G, Doran E, Lott IT, Head E, Cotman CW, Wallace DC. Systemic mitochondrial dysfunction and the etiology of Alzheimer's disease and down syndrome dementia. J Alzheimers Dis. 2010;20(Suppl 2):S293–S310. doi: 10.3233/JAD-2010-100351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ogawa O, Perry G, Smith MA. The "Down's" side of mitochondria. Dev Cell. 2002;2:255–256. doi: 10.1016/s1534-5807(02)00139-9. [DOI] [PubMed] [Google Scholar]
  • 19.Roat E, Prada N, Ferraresi R, Giovenzana C, Nasi M, Troiano L, Pinti M, Nemes E, Lugli E, Biagioni O, Mariotti M, Ciacci L, Consolo U, Balli F, Cossarizza A. Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome. FEBS Lett. 2007;581:521–525. doi: 10.1016/j.febslet.2006.12.058. [DOI] [PubMed] [Google Scholar]
  • 20.Arbuzova S, Hutchin T, Cuckle H. Mitochondrial dysfunction and Down's syndrome. Bioessays. 2002;24:681–684. doi: 10.1002/bies.10138. [DOI] [PubMed] [Google Scholar]
  • 21.Infantino V, Castegna A, Iacobazzi F, Spera I, Scala I, Andria G, Iacobazzi V. Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome. Mol Genet Metab. 2011;102:378–382. doi: 10.1016/j.ymgme.2010.11.166. [DOI] [PubMed] [Google Scholar]
  • 22.Coskun PE, Busciglio J. Oxidative Stress and Mitochondrial Dysfunction in Down’s Syndrome: Relevance to Aging and Dementia. Current Gerontology and Geriatrics Research. 2012;2012:7. doi: 10.1155/2012/383170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Pallardo FV, Lloret A, Lebel M, d'Ischia M, Cogger VC, Le Couteur DG, Gadaleta MN, Castello G, Pagano G. Mitochondrial dysfunction in some oxidative stress-related genetic diseases: Ataxia-Telangiectasia, Down Syndrome. Fanconi Anaemia and Werner Syndrome, Biogerontology. 2010;11:401–419. doi: 10.1007/s10522-010-9269-4. [DOI] [PubMed] [Google Scholar]
  • 24.Mitra K, Lippincott-Schwartz J. Analysis of mitochondrial dynamics and functions using imaging approaches, Current protocols in cell biology / editorial board. Juan S. Bonifacino ... [et al.] 2010;Chapter 4(Unit 4 25 21-21) doi: 10.1002/0471143030.cb0425s46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kalabus JL, Sanborn CC, Jamil RG, Cheng QY, Blanco JG. Expression of the Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 in Hearts from Donors with Down Syndrome. Drug Metabolism and Disposition. 2010;38:2096–2099. doi: 10.1124/dmd.110.035550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Quinones-Lombrana A, Ferguson D, Hageman Blair R, Kalabus JL, Redzematovic A, Blanco JG. Interindividual Variability in the Cardiac Expression of Anthracycline Reductases in Donors With and Without Down Syndrome. Pharmaceutical research. 2014 doi: 10.1007/s11095-013-1267-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–622. doi: 10.1373/clinchem.2008.112797. [DOI] [PubMed] [Google Scholar]
  • 28.Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nature protocols. 2008;3:1101–1108. doi: 10.1038/nprot.2008.73. [DOI] [PubMed] [Google Scholar]
  • 29.Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R. Fast and effective prediction of microRNA/target duplexes. RNA. 2004;10:1507–1517. doi: 10.1261/rna.5248604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Loeb GB, Khan AA, Canner D, Hiatt JB, Shendure J, Darnell RB, Leslie CS, Rudensky AY. Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting. Mol Cell. 2012;48:760–770. doi: 10.1016/j.molcel.2012.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Chen Y, Siegel F, Kipschull S, Haas B, Frohlich H, Meister G, Pfeifer A. miR-155 regulates differentiation of brown and beige adipocytes via a bistable circuit. Nature communications. 2013;4:1769. doi: 10.1038/ncomms2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Piccoli C, Izzo A, Scrima R, Bonfiglio F, Manco R, Negri R, Quarato G, Cela O, Ripoli M, Prisco M, Gentile F, Cali G, Pinton P, Conti A, Nitsch L, Capitanio N. Chronic prooxidative state and mitochondrial dysfunctions are more pronounced in fibroblasts from Down syndrome foeti with congenital heart defects. Human molecular genetics. 2013;22:1218–1232. doi: 10.1093/hmg/dds529. [DOI] [PubMed] [Google Scholar]

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