miR-142 induces accumulation of reactive oxygen species (ROS) by inhibiting pexophagy in aged bone marrow mesenchymal stem cells

Houri, Kei; Mori, Tatsufumi; Onodera, Yuta; Tsujimoto, Takatoshi; Takehara, Toshiyuki; Nakao, Shinichi; Teramura, Takeshi; Fukuda, Kanji

doi:10.1038/s41598-020-60346-2

Download PDF

Article
Open access
Published: 28 February 2020

miR-142 induces accumulation of reactive oxygen species (ROS) by inhibiting pexophagy in aged bone marrow mesenchymal stem cells

Kei Houri¹,
Tatsufumi Mori²,
Yuta Onodera ORCID: orcid.org/0000-0002-0152-6560³,
Takatoshi Tsujimoto¹,
Toshiyuki Takehara³,
Shinichi Nakao¹,
Takeshi Teramura³ &
…
Kanji Fukuda³

Scientific Reports volume 10, Article number: 3735 (2020) Cite this article

2733 Accesses
20 Citations
2 Altmetric
Metrics details

Subjects

Abstract

Elevation of the levels of reactive oxygen species (ROS) is a major tissue-degenerative phenomenon involved in aging and aging-related diseases. The detailed mechanisms underlying aging-related ROS generation remain unclear. Presently, the expression of microRNA (miR)-142-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMMSCs) of aged mice. Overexpression of miR-142 and subsequent observation revealed that miR-142 involved ROS accumulation through the disruption of selective autophagy for peroxisomes (pexophagy). Mechanistically, attenuation of acetyltransferase Ep300 triggered the upregulation of miR-142 in aged BMMSCs, and miR-142 targeted endothelial PAS domain protein 1 (Epas1) was identified as a regulatory protein of pexophagy. These findings support a novel molecular mechanism relating aging-associated ROS generation and organelle degradation in BMMSCs, and suggest a potential therapeutic target for aging-associated disorders that are accompanied by stem cell degeneration.

Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6

Article Open access 15 December 2020

PINK1/Parkin-mediated mitophagy inhibits osteoblast apoptosis induced by advanced oxidation protein products

Article Open access 07 February 2023

Age-related noncanonical TRMT6–TRMT61A signaling impairs hematopoietic stem cells

Article 17 January 2024

Introduction

Aging worsens functions of human tissues and organs at multiple levels, causing a gradual reduction in the ability to resist stress, damage, and various related diseases. Cellular senescence is considered an important aging hallmark and the direct reason for the above mentioned changes^1,2. In recent years, accumulating evidence has indicated that reactive oxygen species (ROS), which include superoxide anion and hydroxyl radicals, generated from both intrinsic and extrinsic events induce cell damage and senescence during aging. There are numerous studies, which report that ROS and oxidative damage increase with age^3,4, that reducing oxidative damage extends the lifespan of various model organisms, and increased production of ROS shortens their lifespan⁵. ROS contribute to cellular senescence onset and progression by damaging mitochondrial DNA (mtDNA) and modifying the telomerase reverse transcriptase (TERT) enzyme⁶, histones, and DNA by acting in interconnected epigenetic phases^7,8. Furthermore, high ROS levels provoke p53 activation, which induces p53-mediated apoptosis and cell senescence⁹. Although all cells in an organism can be affected by the accumulation of ROS, the effects of ROS on stem cells are particularly important for understanding the processes of aging and its related diseases^10,11. Accumulation of oxidative damage in stem cells can lead to loss of stemness, cell transformation, tumorigenesis, or tissue injury¹¹. Thus, elucidating the molecular mechanisms underlying ROS accumulation in stem cells is important to develop therapies for inhibiting the underlying cause of aging-related tissue dysfunction or diseases.

Degenerated cellular organelles are a major source of ROS. Oxygen is consumed in various metabolic reactions in different intracellular locations, with mitochondria, ER, and peroxisomes being the major sites; thus, dysfunction of these organelles directly leads to the generation of a large amount of ROS¹². Among these, the peroxisome is a very important source of ROS, which is mainly produced through metabolic pathways, including fatty acid β-oxidation, photorespiration, nucleic acid, and polyamine catabolism^13,14,15. It has been estimated that about 35% of all H₂O₂ formed in the rat liver is derived from its peroxisomes. The number, morphology, and size of peroxisomes are dynamically regulated in response to environmental and developmental cues¹⁶. Selective autophagy for peroxisomes, called pexophagy, is a quality control mechanism to maintain the proper function of peroxisomes¹⁵. However, the decline of autophagic activity and accumulation of damaged macromolecules and organelles are well known characteristics of aged cells^17,18,19. Importantly, increases in peroxisome number,²⁰ along with reduction of peroxisomal enzymes^20,21 during aging, has also been reported. Although partial mechanisms inducing the decreased expression of genes associated with autophagy have been discovered²², the mechanisms underlying the impairment of autophagic function by aging remain poorly understood.

Recently, short strand noncoding RNAs called miRNAs have been found to be involved in the regulation of autophagy²³. During aging, it has been reported that certain miRNAs are involved in aging-associated degenerative changes, including inhibition of autophagy and promotion of ROS generation^24,25,26.

Here we identified miR-142-5p (miR-142) as a prominent miRNA in aged BMMSCs and found that miR-142 induced increasing numbers of peroxisomes and cellular ROS levels by inhibiting pexophagy through the suppression of Epas1 expression.

Results

To identify miRNAs upregulated in aged BMMSCs, we performed miRNA-sequencing for PαS-double positive cells collected from the bone-marrow (BM) of young and aged mice. It was revealed that 32 miRNAs exhibited a log2 fold change of >3 in the aged BMMSCs. Among these, we chose miRNAs showing a mean value of relative expression >1,000 and focused on miR-142 since it showed the largest difference in expression levels between young and aged BMMSCs (Table S1), and its expression level was higher than ubiquitously expressed miRNA in undifferentiated BMMSCs such as miR-125b²⁷, miR-204²⁸, and let7c²⁹. Quantitative RT-PCR (qRT-PCR) showed the upregulation of miR-142 in the BM and in BMMSCs collected from aged mice (Figs. 1 and S1).

Next, we examined if miR-142 was involved in ROS generation in BMMSCs. When BMMSCs were transfected with a miR-142 mimic, ROS levels increased (Fig. 2A). To determine the reason for the increased ROS levels, we performed electron-microscopic observation of BMMSCs transfected with the miR-142 mimic. Using TEM, we found that the number of peroxisomes increased in BMMSCs containing the miR-142 mimic (Fig. 2B,C). From these results, we hypothesized that miR-142 induced ROS generation via induction of peroxisome accumulation.

As a regulation mechanism of peroxisomal abundance, we focused on pexophagy, which is one of the most important regulatory mechanisms of peroxisomes. To detect peroxisome and pexophagic activity, we produced transgenic cells expressing peroxisome-targeted DsRed (DsRed-PTS1) and LC3B-GFP reflecting autophagy. Under normal conditions, approximately 33% of DsRed-positive peroxisomes overlapped with GFP fluorescence, indicating that these peroxisomes underwent pexophagy. When the cells were observed 1 h after rapamycin treatment, the abundance of GFP/DsRed-double-positive particles increased, while the DsRed single positive particles slightly decreased (Fig. S2). When the cells were treated with Wy-14643, which is a selective agonist of peroxisome proliferator-activated receptor-α³⁰, the abundance of DsRed positive particles significantly increased (Fig. S2). Based on these results, we concluded that our system was useful for detecting both peroxisomes and pexophagy. By transfecting the miR-142 mimic, the abundance of DsRed-positive particles representing peroxisomes significantly increased. However, GFP signals that represented autophagy were suppressed in the cells (Fig. 3A,B). To determine whether the pexophagy activity was affected by miR-142, we produced a pexophagy reporter that is a GFP and PTS1-tagged tandem monomeric DsRed (RFP). The reporter protein locates to the peroxisomes by the PTS1 peroxisome targeting signal, and when an autophagosome targets the peroxisome, the GFP domain of the reporter is digested by lysosomes and the DsRed fragment, which is relatively stable, remained³¹. Under autophagy-induced culture conditions, a clear band showing the DsRed domain was detected. Conversely, the DsRed fragments were decreased by transfection of miR-142 mimic (Fig. 3C)

To elucidate the mechanism of the miR-142-mediated pexophagy regulation, we performed target prediction using miRDB³² and DIANA-Tarbase v 7.0³³. From the results of the prediction analysis and previous studies³⁴, we hypothesized that miR-142 affects peroxisome abundance through suppression of Epas1, which was an important regulator of pexophagy, in aged BMMSCs. To demonstrate that Epas1 plays an important role in pexophagy during aging, we observed the expression level of Epas1 in young and aged BMMSCs, and then performed siRNA-mediated suppression of Epas1 to evaluate pexophagy activity using the DsRed-PTS1/LC3-GFP system in BMMSCs. In the aged BMMSCs, Epas1 expression was suppressed compared with young BMMSCs (Fig. 4A). Treatment with siRNA against Epas1 (siEpas1) resulted in approximately 85% suppression of Epas1 mRNA compared with control treated with a scrambled siRNA sequence and ROS generation (Figs. 4B and S3). Western blot (WB) analysis showed that decreased Epas1 expression also occurred at the protein level with siRNA treatment (Fig. 4C). The decreased amount of Epas1 affected the pexophagy-related genes Pex10 and Pex14. Consistent with previous studies³⁴, peroxisomal abundance increased and LC3-GFP-positive peroxisomes showing pexophagy decreased under siEpas1 conditions (Figs. 4D and S3). Consistently, the GFP-DsRed tandem reporter assay also shows that the reduction of Epas1 expression suppressed pexophagy activity (Fig. 4E).

We investigated if miR-142 downregulated Epas1 expression in BMMSCs. As expected, the expression level of Epas1 was suppressed in BMMSCs transfected with the miR-142 mimic (Fig. 5A). WB analysis also showed decreased expression of Epas1 in BMMSCs transfected with the miR-142 mimic (Fig. 5B). To confirm if miR-142 targets Epas1, we constructed a luciferase (Luc) expression plasmid containing the 3′ UTR sequence of Epas1 and compared the expression levels between wild-type Luc and Luc-Epas1 3′ UTR in BMMSCs transfected with the miR-142 mimic. In normal BMMSCs, the expression levels of Luc were not different either with or without Epas1 3′ UTR. In contrast, expression of Luc-Epas1 3′ UTR was significantly suppressed in BMMSCs transfected with the miR-142 mimic (Fig. 5C). To further determine the molecular relationship between miR-142, Epas1, and pexophagy, we performed a compensation experiment using an overexpression plasmid for an active mutant of Epas1 (acEpas1), which is stable even under normoxia and lacks a 3′ UTR³⁵. Consistent with our hypothesis, the introduction of acEpas1 restored pexophagy activity and suppressed the miR-142-induced peroxisomal accumulation (Fig. 5D,E), indicating that the miR-142-mediated suppression of pexophagy occurred via regulation of Epas1. To determine whether the suppression of pexophagy by miR-142 was canceled by overexpression of acEpas1, we performed the GFP-DsRed tandem reporter assay and observed that pexophagy activity was recovered by acEpas1 expression (Fig. 5F).

Next, we examined the molecular mechanisms responsible for upregulation of miR-142 in aged tissues. It has been demonstrated that the expression level of miR-142 is suppressed by acetyltransferase Ep300³⁶. Ep300 is a protein that undergoes aging-associated downregulation³⁷. We found that the expression level of Ep300 was lower in aged BM and BMMSCs than in young mice (Figs. 6A and S1). From these data, we hypothesized that suppression of Ep300 increased expression of miR-142 in aged BMMSCs. To validate this hypothesis, we suppressed expression of Ep300 with a siRNA in BMMSCs and observed the expression levels of miR-142 and Epas1. When the BMMSCs were treated with siRNA against Ep300 (siEp300), a significant decrease in Ep300 (Fig. 6B,C), an increase in miR-142, and a decrease in Epas1 expression were observed (Fig. 6D). In the siEP300 treated cells, amounts of ROS levels, accumulation of peroxisomes, and amounts of pexophagy were examined. Transfection of siEP300 increased intra-cellular ROS levels (Fig. 6E). The effect of siEp300 and miR-142 was additive; both the fluorescence imaging assay with the DsRed-PTS1/LC3-GFP system and the GFP-DsRed tandem reporter assay showed that co-transfection of the miR-142 mimic and siEP300 increased suppression of pexophagy compared with the single transfection of miR-142 (Fig. 6F,G).

From these results, we concluded that miR-142 received suppressive regulation by Ep300 and that disruption of Ep300 during aging could be a trigger for the upregulation of miR-142 expression and the subsequent disruption of pexophagy regulation and ROS generation in aged cells.

Discussion

Accumulated ROS disturbs the maintenance and proliferative ability of stem cells³⁸ by activating cell cycle suppressors³⁹ or inducing differentiation commitment^40,41. Furthermore, elevated ROS levels in MSCs reduce their engraftment potential and induce apoptosis after transplantation^26,42. Thus, elucidating the molecular mechanisms underlying ROS accumulation and the development of therapeutic ROS modulation are essential to address aging and aging-related diseases. As a molecule involved in ROS accumulation in aged stem cells, we focused on the aging-associated miRNAs and performed miRNA sequencing. The results revealed that miR-142 was significantly upregulated in aged BMMSCs.

Consistent with our observation, Park et al., also found that the expression level of miR-142-5p increases in dendritic cells derived from aged bone marrow⁴³. However, Zang et al. reported that the expression level of miR-142 in serum decreases during the aging process⁴⁴. Tissue or cell lineage specificity could be responsible for the discrepancy. For example, Zang et al. observed decreased expression of miR-29b in serum obtained from aged individuals. However, miR-29b is upregulated in aged brain⁴⁵, aorta⁴⁶, and cochlear tissues⁴⁷. These findings highlight that care is needed in the selection of miR-142 as a marker for detection of tissue/organ aging or as a common molecule inducing stem cell aging in different tissues.

In the cultured BMMSCs transfected with miR-142, an increased ROS level was detected with the CellROX assay. Previously, several research groups have demonstrated the involvement of miRNA in ROS generation. For example, miR-210, which is upregulated during hypoxia, induces accumulation of ROS and apoptosis⁴⁸ by suppressing mitochondrial activity⁴⁹. miR-142⁵⁰, miR-377⁵¹ and miR-200c⁵² are also reported to be involved in ROS generation by targeting anti-oxidant genes. However, suppression of antioxidants such as Nrf2, Sod1, Foxo1, and Foxo3 by miR-142 was not significant in the present study (Fig. S4). In a previous study, we addressed a mechanism of the aging-associated miRNA (Ag-miRNA) induced ROS generation and revealed that miR-155 inhibits expression of antioxidant related genes and increased ROS levels by debilitating tolerance against oxidation stresses. We hypothesized that miR-142 induced ROS accumulation through a considerably different mechanism from the previous models since overexpression of miR-142 further exacerbated the miR-155-induced ROS accumulation (Fig. S5). Interestingly, TEM observation clearly revealed that the number of peroxisomes increased in BMMSCs transfected with the miR-142 mimic. This phenomenon was reproducible even when unpassaged primary BMMSCs with short culture period (120 h) were used (Fig. S6).

Under normal physiological conditions, the amount of peroxisomes is regulated by a balance between biogenesis and turnover. Especially, the selective autophagy of peroxisomes—pexophagy—is an important process responsible for the maintenance of peroxisomal quality and quantity⁵³. Senescent cells contain approximately twice as many peroxisomes immunolabeled with the peroxisomal membrane protein 70 kDa (PMP70) or Pex14p²⁰, as compared to early passage cells. Similarly, peroxisomes proliferate in the human retinal epithelia of aged individuals⁵⁴. These findings indicate that the turnover of peroxisomes is disrupted in aged cells/tissues, and could be a cause of ROS generation that occurs with aging. In our study with a transgenic BMMSC line stably expressing LC3-GFP/DsRed-PTS1, we hypothesized that miR-142 suppressed pexophagy and led to increased numbers of peroxisomes. To date, it has been posited that a ubiquitin-binding autophagic receptor Nbr1 is necessary and sufficient for pexophagy activity^34,55. On the other hand, miR-142 did not seem to affect the gene expression of Nbr1 at least in our study. Compared with the direct suppression of Nbr1 by siRNA, in which a three-fold increase in peroxisomes compared with control was detected, the effect of miR-142 overexpression for peroxisome accumulation was weak. However, the function of miR-142 was additive to siNbr1 treatment (Figs. S7 and S8). These findings indicate that miR-142 regulates pexophagy by targeting an upstream or independent molecule from Nbr1. We investigated the direct targets of miR-142 using target prediction with open software, and focused on Epas1 (Hypoxia inducible factor 2A), which is a master regulator of the adaptive response to hypoxia. Walter et al. reported that hypoxia inducible factor (HIF) 2A ensures the efficient depletion of the peroxisome pool by simultaneously inducing pexophagy³⁴. As a possible reason for the regulation of pexophagy by Epas1, peroxisomal function may be highly dependent on oxygen concentration⁵⁶. Thus HIF signaling that is used to adapt to low oxygen conditions could be a suitable molecule to regulate the numbers of peroxisomes and their oxygen consumption. Although the aging-dependent expression changes of Epas1 in BMMSCs are still unclear, significant downregulation of Epas1 in aged PαS BMMSCs were observed in the present study. Evidence supporting the idea that Epas1 was reduced in aged BM cells was also found in a previous gene expression profile⁵⁷. These data prompted our hypothesis that Epas1 could be a promising candidate mediating miR-142 expression and the dysregulation of pexophagy in aged BMMSCs. Consistent with a previous report³⁴, suppression of Epas1 resulted in deterioration of pexophagy and accumulation of peroxisomes. Furthermore, consistent with our expectation, Epas1 expression was suppressed in BMMSCs transfected with the miR-142 mimic. However, overexpression of Epas1 counteracted the miR-142 induced proliferation of peroxisomes. From these results, we concluded that miR-142 suppressed pexophagy via regulation of Epas1.

As a factor triggering the upregulation of miR-142, we focused on Ep300. It has been reported that miR-142 is downregulated by acetyltransferase Ep300³⁶. Importantly, Ep300 appears to be downregulated with aging³⁷ and with increasing doubling times in cultured cells⁵⁸. Furthermore, it has also been reported that inhibition of Ep300 can induce aging phenotypes, such as senescence associated β-galactosidase expression⁵⁸ and accumulation of DNA damage⁵⁹. Consistent with our expectation, suppression of Ep300 led to increased expression of miR-142, accumulation of peroxisomes, and accumulation of ROS. These data support the conclusion that there is a molecular pathway that is responsible for inducing ROS generation in aged BMMSCs. In the first step of this pathway, reduction of Ep300, which occurs during aging or cellular senescence processes, leads to upregulation of miR-142. The increased miR-142 targets Epas1, and the reduced level of Epas1 results in disruption of the normal maintenance of peroxisomes (Fig. 7) . From the perspective of a therapeutic application, miR-142 could be an attractive target for miRNA-targeting therapy, since miR-142 is highly expressed in Alzheimer’s disease⁶⁰, multiple sclerosis⁶¹, and osteoporosis⁶², in addition to aged tissues. These findings indicated that the inhibition of miR-142 using a specific inhibitor could be a potential therapeutic approach for some chronic diseases and various aging-associated cell/tissue degenerations.

Obviously, it is impossible to explain the aging-associated deterioration of peroxisomal maintenance and ROS generation only by the above scheme. We think there are at least three limitations. First, reduction of Ep300 and Epas1 may possibly generate ROS without mediation of miR-142. For example, Sun et al., reported that acetylation of Nrf2 by CBP/Ep300 involves maximum binding of Nrf2 to specific ARE-containing promoters⁶³. This suggests that the reduction of Ep300 could lead to the wasting of anti-oxidation-related gene expression. Furthermore, several studies showed that depletion of Epas1 expression can reduce the expression of genes related to anti-oxidant systems, such as Sod1, catalase, and heme oxygenase 1 (Hmox1)^64,65. Second, miR-142 expression can be enhanced not only by reduction of Ep300 but also by stimulation with enhancer molecules. The inflammatory cytokines, IL-4 and IL-13, can trigger miR-142 expression⁶⁶. It is well known that aging tissues are in an inflammation-like condition and express various inflammatory molecules. Therefore, it is highly possible that these cytokine signals and Ep300 downregulation simultaneously enhance expression of miR-142 in the actual tissues where aging-associated degeneration occurs. Third, we should pay attention to the roles of miR-142 in normal development and tissue homeostasis. In general, miRNAs have multiple target genes. Thus, understanding its original role is very complicated. For example, even miR-21, which is one of the well-studied pathogenic miRNAs, is important for normal development^67,68; it has been reported that miR-142 is critical for hematopoiesis⁶⁹ and the control of adaptive growth in cardiomyocytes⁷⁰. Thus, further detailed studies of the targets and functions of miR-142 in normal tissue homeostasis are essential. This understanding may enable the use of miR-142 as a marker and target for therapeutic control.

Materials and Methods

Ethical statement

All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Kindai University. All experiments using animals were performed in accordance with institutional guidelines and regulations. IACUC approved project No. is KAME-26-043.

Isolation of the MSC from BM tissues (BMMSCs)

Four-week- and 1.5-year-old C57BL/6N male mice were used for the experiment as young and aged model. Bone marrow tissues were prepared as previously reported⁷¹. Long bones were collected from the hind limbs of the euthanized mice. The bone marrow was flushed out with α-MEM twice, and the residual long bones were cut into small pieces around 2–3 mm³ and treated with collagenase type II for 15 min. Then, dissociated tissues were washed twice with PBS and reacted with anti-PDGFRα (17-1401-81, eBioscience, San Diego, CA, USA), anti-Sca1 (61-5981-82, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD45 (35-0451-U500, TONBO bioscience, San Diego, CA, USA), and anti-Ter119 (35-5921-U500, TONBO bioscience) for FACS isolation of the MSCs. As a control, cells were reacted with isotype IgG conjugated with each florescent dye (eBioscience). The PDGFRα+/Sca1+/CD45−/Ter119− population was sorted using a FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA).

Detection of ROS in the BMMSCs from young and aged mice

BMMSCs were prepared as described above. ROS levels were analyzed using the CellROX Green Reagent (Thermo Fisher Scientific) and a FACS Canto^TM II (BD Biosciences).

miRNA sequence

Libraries were generated from 10–20 ng of RNA, which was prepared from the FACS-sorted PDGFRα+/Sca1+/CD45−/Ter119− cells with the miRNeasy Micro Kit (Qiagen, Hilden, Germany), using the SMARTer smRNA-seq Kit (TAKARA Bio Inc., Shiga, Japan). The next-generation single-read sequencing was performed using the sequencer NextSeq500 (Illumina, San Diego, CA, USA) using the single-end 50 nt high output sequencing mode. The trimmed reads were mapped to the reference genome of mouse (GRCm38.p5) by STAR (version 2.6.1a) program. The sequence quality was assessed with FastQC software.

Preparation of mouse BMMSCs

BMMSCs were isolated as previously reported²⁶. BMMSCs were cultured in α-MEM (Wako, Tokyo, Japan) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) under 5% CO₂ and 5% O₂ at 37 °C.

Fluorescent observation of pexophagy

A cDNA encoding the monomeric derivative of DsRed fluorescent protein was cloned into the piggybac cDNA expression vector with the PTS1 (serine-lysine-leucine) sequence⁷² using the In-Fusion HD Cloning Kit (TAKARA Bio Inc., Shiga, Japan).

The LC3-GFP cDNA was amplified from the pEGFP-LC3 plasmid (Addgene #21073) using the Tks Gflex DNA polymerase (TAKARA Bio Inc.) and cloned into the piggybac cDNA expression vector. PTS1-DsRed and pEGFP-LC3 was introduced using a CUY21 electroporator (NEPA Gene, Tokyo, Japan). Fluorescent images were captured using a BZ-X710 all-in-one fluorescent microscope (KEYENCE Corporation, Osaka, Japan) and analyzed integrated values of fluorescent brightness for each channel by an image analysis software BZ-X Analyzer Ver 1.3 (KEYENCE Corporation). To determine whether each fluorescent protein localized to peroxisomes and autophagosomes, a chemical activator of peroxisome proliferation, Wy14643³⁰ and that of autophagy, Rapamycin was used⁷³. DsRed/GFP double-positive dots were counted as peroxisomes processed by autophagy (pexophagy).

Pexophagy activity detection assay

The EGFP sequence was amplified using Tks Gflex DNA polymerase and subcloned into the Nco I restriction enzyme site with a GGC short linker using the In-Fusion HD Cloning system. To enhance pexophagy activity, the BMMSCs were cultured in the amino acid-free DMEM for autophagy induction (Fujifilm Holdings Corporation, Tokyo, Japan) for 1 h before sampling. The full length and digested reporter molecules were detected using anti-RFP antibody (αRFP, M204-3, MBL, Nagoya, Japan) by WB as detailed below.

RT-PCR for miRNA

RNA preparation and cDNA production were performed using the TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA) and the miRNA cDNA Synthesis Kit with the Poly(A) Polymerase Tailing Kit (Applied Biological Materials Inc., Rochmond, BC, USA). Quantitative RT-PCR was performed using BrightGreen Express 2X qPCR MasterMix-No Dye (Applied Biological Materials Inc., BC, Canada) with a Thermal Cycler Dice® Real Time System at 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 60 °C for 15 s. Expression quantity was evaluated by the ΔΔCt method (ΔΔCt = ΔCt_sample − ΔCt_control). U6 snRNA was used as a control gene for normalization.

RT-PCR for mRNA

RNA isolation and subsequent reverse-transcription were performed using TRI Reagent® and the PrimeScript® RT Master Mix Kit (TAKARA Bio Inc.). Quantitative real-time PCR was performed using Perfect real-time SYBR green II (TAKARA Bio Inc.) and a Thermal Cycler Dice® Real Time System. Expression quantity was evaluated by the ΔΔCt method using Gapdh as a control gene for normalization. To prevent contamination of genomic DNA, primers were designed to span at least one intron. Primer sequences are listed in Supplementary Table S2.

WB analysis

All samples were lysed in SDS buffer, electroporated and blotted onto a PVDF membrane (Hybond-P; GE Healthcare Japan, Tokyo, Japan). The PVDF membranes were then treated with Block Ace for 1 hour (Dainippon Sumitomo Pharma, Osaka, Japan) and reacted with primary antibodies overnight at 4 °C. Chemiluminescence detection was performed with horseradish peroxidase (HRP)-conjugated secondary antibodies and Immunostar^® LD (Wako) reagents. Antibodies are listed in Supplementary Table S3. The raw WB data are shown in Supplementary Fig. S9.

Transfection of the mimic RNA and siRNA into mouse BMMSCs

Mimic RNA of miR-142 (mmu-miR142a-5p mimic, GeneDesign, Inc., Osaka, Japan) and siRNA against Epas1 or Ep300 were transfected using the Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Sequences of the siRNAs used are shown in Supplementary Table S4.

Transmission electron microscopy (TEM) observation

BMMSCs were fixed in 2% glutaraldehyde in 50 mM PIPES buffer at room temperature for 2.5 h, then post-fixed, following rinsing, in 1% OsO₄ for 1 h. Following dehydration in ethanol, the cell pellets were embedded in epoxy resin, cut into 70 nm sections, stained with uranyl acetate and lead citrate, and viewed with a HT7700 Automated TEM (Hitachi High Technologies, Inc., Tokyo, Japan) at 100 kV.

Statistical analysis

Significant differences were detected with the Tukey-Kramer HSD test when the experiment had >three different groups. The data from two different groups were analyzed with a Student’s t-test, as appropriate. Caliculations were performed using JMP software version 10.0.0 (SAS Institute, Cary, NC, USA). Differences were assessed with a two-sided test with an α level of 0.05. The numbers of replicates (N) indicate biological replicates from independent experiments.

References

Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217, https://doi.org/10.1016/j.cell.2013.05.039 (2013).
Article CAS PubMed PubMed Central Google Scholar
Cruickshanks, H. A. et al. Senescent cells harbour features of the cancer epigenome. Nat. Cell Biol. 15, 1495–1506, https://doi.org/10.1038/ncb2879 (2013).
Article CAS PubMed PubMed Central Google Scholar
Stadtman, E. R. Protein oxidation and aging. Sci. 257, 1220–1224 (1992).
Article ADS CAS Google Scholar
Cui, H., Kong, Y. & Zhang, H. Oxidative stress, mitochondrial dysfunction, and aging. J. Signal. Transduct. 2012, 646354, https://doi.org/10.1155/2012/646354 (2012).
Article CAS PubMed Google Scholar
Wickens, A. P. Ageing and the free radical theory. Respir. Physiol. 128, 379–391 (2001).
Article CAS PubMed Google Scholar
van Deursen, J. M. The role of senescent cells in ageing. Nat. 509, 439–446, https://doi.org/10.1038/nature13193 (2014).
Article ADS CAS Google Scholar
D’Aquila, P., Bellizzi, D. & Passarino, G. Mitochondria in health, aging and diseases: the epigenetic perspective. Biogerontology 16, 569–585, https://doi.org/10.1007/s10522-015-9562-3 (2015).
Article CAS PubMed Google Scholar
Afanas’ev, I. New nucleophilic mechanisms of ros-dependent epigenetic modifications: comparison of aging and cancer. Aging Dis. 5, 52–62, https://doi.org/10.14336/AD.2014.050052 (2014).
Article PubMed Google Scholar
Ahmed, E. K., Rogowska-Wrzesinska, A., Roepstorff, P., Bulteau, A. L. & Friguet, B. Protein modification and replicative senescence of WI-38 human embryonic fibroblasts. Aging Cell 9, 252–272, https://doi.org/10.1111/j.1474-9726.2010.00555.x (2010).
Article CAS PubMed Google Scholar
Kobayashi, C. I. & Suda, T. Regulation of reactive oxygen species in stem cells and cancer stem cells. J. Cell Physiol. 227, 421–430, https://doi.org/10.1002/jcp.22764 (2012).
Article CAS PubMed Google Scholar
Shyh-Chang, N., Daley, G. Q. & Cantley, L. C. Stem cell metabolism in tissue development and aging. Dev. 140, 2535–2547, https://doi.org/10.1242/dev.091777 (2013).
Article CAS Google Scholar
Moldovan, L. & Moldovan, N. I. Oxygen free radicals and redox biology of organelles. Histochem. Cell Biol. 122, 395–412, https://doi.org/10.1007/s00418-004-0676-y (2004).
Article CAS PubMed Google Scholar
Deb, R. & Nagotu, S. Versatility of peroxisomes: An evolving concept. Tissue Cell 49, 209–226, https://doi.org/10.1016/j.tice.2017.03.002 (2017).
Article CAS PubMed Google Scholar
Cho, D. H., Kim, Y. S., Jo, D. S., Choe, S. K. & Jo, E. K. Pexophagy: Molecular Mechanisms and Implications for Health and Diseases. Mol. Cell 41, 55–64, https://doi.org/10.14348/molcells.2018.2245 (2018).
Article CAS Google Scholar
Kumar, S., Kawalek, A. & van der Klei, I. J. Peroxisomal quality control mechanisms. Curr. Opin. Microbiol. 22, 30–37, https://doi.org/10.1016/j.mib.2014.09.009 (2014).
Article CAS PubMed Google Scholar
Heiland, I. & Erdmann, R. Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins. FEBS J. 272, 2362–2372, https://doi.org/10.1111/j.1742-4658.2005.04690.x (2005).
Article CAS PubMed Google Scholar
Terman, A. & Brunk, U. T. Myocyte aging and mitochondrial turnover. Exp. Gerontol. 39, 701–705, https://doi.org/10.1016/j.exger.2004.01.005 (2004).
Article CAS PubMed Google Scholar
Martinez-Lopez, N., Athonvarangkul, D. & Singh, R. Autophagy and aging. Adv. Exp. Med. Biol. 847, 73–87, https://doi.org/10.1007/978-1-4939-2404-2_3 (2015).
Article CAS PubMed PubMed Central Google Scholar
Cuervo, A. M. & Macian, F. Autophagy and the immune function in aging. Curr. Opin. Immunol. 29, 97–104, https://doi.org/10.1016/j.coi.2014.05.006 (2014).
Article CAS PubMed Google Scholar
Legakis, J. E. et al. Peroxisome senescence in human fibroblasts. Mol. Biol. Cell 13, 4243–4255, https://doi.org/10.1091/mbc.e02-06-0322 (2002).
Article CAS PubMed PubMed Central Google Scholar
Perichon, R., Bourre, J. M., Kelly, J. F. & Roth, G. S. The role of peroxisomes in aging. Cell Mol. Life Sci. 54, 641–652, https://doi.org/10.1007/s000180050192 (1998).
Article CAS PubMed Google Scholar
Escobar, K. A., Cole, N. H., Mermier, C. M. & VanDusseldorp, T. A. Autophagy and aging: Maintaining the proteome through exercise and caloric restriction. Aging Cell, e12876, https://doi.org/10.1111/acel.12876 (2018).
Xu, J., Wang, Y., Tan, X. & Jing, H. MicroRNAs in autophagy and their emerging roles in crosstalk with apoptosis. Autophagy 8, 873–882, https://doi.org/10.4161/auto.19629 (2012).
Article CAS PubMed PubMed Central Google Scholar
Harries, L. W. MicroRNAs as Mediators of the Ageing Process. Genes. 5, 656–670, https://doi.org/10.3390/genes5030656 (2014).
Article CAS PubMed PubMed Central Google Scholar
Deshpande, S. et al. Reduced Autophagy by a microRNA-mediated Signaling Cascade in Diabetes-induced Renal Glomerular Hypertrophy. Sci. Rep. 8, 6954, https://doi.org/10.1038/s41598-018-25295-x (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Onodera, Y. et al. miR-155 induces ROS generation through downregulation of antioxidation-related genes in mesenchymal stem cells. Aging Cell 16, 1369–1380, https://doi.org/10.1111/acel.12680 (2017).
Article CAS PubMed PubMed Central Google Scholar
Mizuno, Y. et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem. Biophys. Res. Commun. 368, 267–272, https://doi.org/10.1016/j.bbrc.2008.01.073 (2008).
Article CAS PubMed Google Scholar
Huang, J., Zhao, L., Xing, L. & Chen, D. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cell 28, 357–364, https://doi.org/10.1002/stem.288 (2010).
Article CAS Google Scholar
Chen, T. S. et al. Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res. 38, 215–224, https://doi.org/10.1093/nar/gkp857 (2010).
Article CAS PubMed Google Scholar
Oruqaj, G. et al. Compromised peroxisomes in idiopathic pulmonary fibrosis, a vicious cycle inducing a higher fibrotic response via TGF-beta signaling. Proc. Natl Acad. Sci. USA 112, E2048–2057, https://doi.org/10.1073/pnas.1415111112 (2015).
Article CAS PubMed PubMed Central Google Scholar
Chino, H., Hatta, T., Natsume, T. & Mizushima, N. Intrinsically Disordered Protein TEX264 Mediates ER-phagy. Mol. Cell 74, 909–921 e906, https://doi.org/10.1016/j.molcel.2019.03.033 (2019).
Article CAS PubMed Google Scholar
Wong, N. & Wang, X. miRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res. 43, D146–152, https://doi.org/10.1093/nar/gku1104 (2015).
Article CAS PubMed Google Scholar
Vergoulis, T. et al. TarBase 6.0: capturing the exponential growth of miRNA targets with experimental support. Nucleic Acids Res. 40, D222–229, https://doi.org/10.1093/nar/gkr1161 (2012).
Article CAS PubMed Google Scholar
Walter, K. M. et al. Hif-2alpha promotes degradation of mammalian peroxisomes by selective autophagy. Cell Metab. 20, 882–897, https://doi.org/10.1016/j.cmet.2014.09.017 (2014).
Article CAS PubMed Google Scholar
Hu, C. J., Wang, L. Y., Chodosh, L. A., Keith, B. & Simon, M. C. Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol. Cell Biol. 23, 9361–9374 (2003).
Article CAS PubMed PubMed Central Google Scholar
Sharma, S. et al. Repression of miR-142 by p300 and MAPK is required for survival signalling via gp130 during adaptive hypertrophy. EMBO Mol. Med. 4, 617–632, https://doi.org/10.1002/emmm.201200234 (2012).
Article CAS PubMed PubMed Central Google Scholar
Li, Q., Xiao, H. & Isobe, K. Histone acetyltransferase activities of cAMP-regulated enhancer-binding protein and p300 in tissues of fetal, young, and old mice. J. Gerontol. A Biol. Sci. Med. Sci 57, B93–98 (2002).
Article PubMed Google Scholar
Ito, K. et al. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat. Med. 12, 446–451, https://doi.org/10.1038/nm1388 (2006).
Article CAS PubMed Google Scholar
Ito, K. et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nat. 431, 997–1002, https://doi.org/10.1038/nature02989 (2004).
Article ADS CAS Google Scholar
Lin, C. H., Li, N. T., Cheng, H. S. & Yen, M. L. Oxidative stress induces imbalance of adipogenic/osteoblastic lineage commitment in mesenchymal stem cells through decreasing SIRT1 functions. J. Cell Mol. Med. 22, 786–796, https://doi.org/10.1111/jcmm.13356 (2018).
Article CAS PubMed Google Scholar
Denu, R. A. & Hematti, P. Effects of Oxidative Stress on Mesenchymal Stem Cell Biology. Oxid. Med. Cell Longev. 2016, 2989076, https://doi.org/10.1155/2016/2989076 (2016).
Article CAS PubMed PubMed Central Google Scholar
Rodrigues, M., Turner, O., Stolz, D., Griffith, L. G. & Wells, A. Production of reactive oxygen species by multipotent stromal cells/mesenchymal stem cells upon exposure to fas ligand. Cell Transpl. 21, 2171–2187, https://doi.org/10.3727/096368912X639035 (2012).
Article Google Scholar
Park, S., Kang, S., Min, K. H., Woo Hwang, K. & Min, H. Age-associated changes in microRNA expression in bone marrow derived dendritic cells. Immunol. Invest. 42, 179–190, https://doi.org/10.3109/08820139.2012.717328 (2013).
Article CAS PubMed Google Scholar
Zhang, H. et al. Investigation of microRNA expression in human serum during the aging process. J. Gerontol. A Biol. Sci. Med. Sci 70, 102–109, https://doi.org/10.1093/gerona/glu145 (2015).
Article CAS PubMed Google Scholar
Fenn, A. M. et al. Increased micro-RNA 29b in the aged brain correlates with the reduction of insulin-like growth factor-1 and fractalkine ligand. Neurobiol. Aging 34, 2748–2758, https://doi.org/10.1016/j.neurobiolaging.2013.06.007 (2013).
Article CAS PubMed PubMed Central Google Scholar
Boon, R. A. et al. MicroRNA-29 in aortic dilation: implications for aneurysm formation. Circ. Res. 109, 1115–1119, https://doi.org/10.1161/CIRCRESAHA.111.255737 (2011).
Article CAS PubMed Google Scholar
Xue, T. et al. miR-29b overexpression induces cochlear hair cell apoptosis through the regulation of SIRT1/PGC-1alpha signaling: Implications for age-related hearing loss. Int. J. Mol. Med. 38, 1387–1394, https://doi.org/10.3892/ijmm.2016.2735 (2016).
Article CAS PubMed PubMed Central Google Scholar
Chan, S. Y. et al. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metab. 10, 273–284, https://doi.org/10.1016/j.cmet.2009.08.015 (2009).
Article CAS PubMed PubMed Central Google Scholar
Chen, Z., Li, Y., Zhang, H., Huang, P. & Luthra, R. Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression. Oncogene 29, 4362–4368, https://doi.org/10.1038/onc.2010.193 (2010).
Article CAS PubMed Google Scholar
Wang, N. et al. Down-regulation of microRNA-142-5p attenuates oxygen-glucose deprivation and reoxygenation-induced neuron injury through up-regulating Nrf2/ARE signaling pathway. Biomed. Pharmacother. 89, 1187–1195, https://doi.org/10.1016/j.biopha.2017.03.011 (2017).
Article CAS PubMed Google Scholar
Wang, Q. et al. MicroRNA-377 is up-regulated and can lead to increased fibronectin production in diabetic nephropathy. FASEB J. 22, 4126–4135, https://doi.org/10.1096/fj.08-112326 (2008).
Article CAS PubMed PubMed Central Google Scholar
Carlomosti, F. et al. Oxidative Stress-Induced miR-200c Disrupts the Regulatory Loop Among SIRT1, FOXO1, and eNOS. Antioxid. Redox Signal. 27, 328–344, https://doi.org/10.1089/ars.2016.6643 (2017).
Article CAS PubMed Google Scholar
Anding, A. L. & Baehrecke, E. H. Cleaning House: Selective Autophagy of Organelles. Dev. Cell 41, 10–22, https://doi.org/10.1016/j.devcel.2017.02.016 (2017).
Article CAS PubMed PubMed Central Google Scholar
Feher, J. et al. Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration. Neurobiol. Aging 27, 983–993, https://doi.org/10.1016/j.neurobiolaging.2005.05.012 (2006).
Article CAS PubMed Google Scholar
Deosaran, E. et al. NBR1 acts as an autophagy receptor for peroxisomes. J. Cell Sci. 126, 939–952, https://doi.org/10.1242/jcs.114819 (2013).
Article CAS PubMed Google Scholar
De Duve, C. & Baudhuin, P. Peroxisomes (microbodies and related particles). Physiol. Rev. 46, 323–357, https://doi.org/10.1152/physrev.1966.46.2.323 (1966).
Article PubMed Google Scholar
Liu, L. F., Shen, W. J., Ueno, M., Patel, S. & Kraemer, F. B. Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes. BMC Genomics 12, 212, https://doi.org/10.1186/1471-2164-12-212 (2011).
Article CAS PubMed PubMed Central Google Scholar
Bandyopadhyay, D. et al. Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes. Cancer Res. 62, 6231–6239 (2002).
CAS PubMed Google Scholar
Yan, G. et al. Selective inhibition of p300 HAT blocks cell cycle progression, induces cellular senescence, and inhibits the DNA damage response in melanoma cells. J. Invest. Dermatol. 133, 2444–2452, https://doi.org/10.1038/jid.2013.187 (2013).
Article CAS PubMed PubMed Central Google Scholar
Song, J. & Kim, Y. K. Identification of the Role of miR-142-5p in Alzheimer’s Disease by Comparative Bioinformatics and Cellular Analysis. Front. Mol. Neurosci. 10, 227, https://doi.org/10.3389/fnmol.2017.00227 (2017).
Article CAS PubMed PubMed Central Google Scholar
Talebi, F. et al. MicroRNA-142 regulates inflammation and T cell differentiation in an animal model of multiple sclerosis. J. Neuroinflammation 14, 55, https://doi.org/10.1186/s12974-017-0832-7 (2017).
Article CAS PubMed PubMed Central Google Scholar
Teng, Z. et al. miR-142-5p in Bone Marrow-Derived Mesenchymal Stem Cells Promotes Osteoporosis Involving Targeting Adhesion Molecule VCAM-1 and Inhibiting Cell Migration. Biomed. Res. Int. 2018, 3274641, https://doi.org/10.1155/2018/3274641 (2018).
Article CAS PubMed PubMed Central Google Scholar
Sun, Z., Chin, Y. E. & Zhang, D. D. Acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 during the antioxidant response. Mol. Cell Biol. 29, 2658–2672, https://doi.org/10.1128/MCB.01639-08 (2009).
Article CAS PubMed PubMed Central Google Scholar
Bertout, J. A., Patel, S. A. & Simon, M. C. The impact of O2 availability on human cancer. Nat. Rev. Cancer 8, 967–975, https://doi.org/10.1038/nrc2540 (2008).
Article CAS PubMed PubMed Central Google Scholar
Majmundar, A. J., Wong, W. J. & Simon, M. C. Hypoxia-inducible factors and the response to hypoxic stress. Mol. Cell 40, 294–309, https://doi.org/10.1016/j.molcel.2010.09.022 (2010).
Article CAS PubMed PubMed Central Google Scholar
Su, S. et al. miR-142-5p and miR-130a-3p are regulated by IL-4 and IL-13 and control profibrogenic macrophage program. Nat. Commun. 6, 8523, https://doi.org/10.1038/ncomms9523 (2015).
Article ADS CAS PubMed Google Scholar
Zhu, S. et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 18, 350–359, https://doi.org/10.1038/cr.2008.24 (2008).
Article CAS PubMed Google Scholar
Cheng, Y. & Zhang, C. MicroRNA-21 in cardiovascular disease. J. Cardiovasc. Transl. Res. 3, 251–255, https://doi.org/10.1007/s12265-010-9169-7 (2010).
Article PubMed PubMed Central Google Scholar
Chapnik, E. et al. miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis. Elife 3, e01964, https://doi.org/10.7554/eLife.01964 (2014).
Article CAS PubMed PubMed Central Google Scholar
Shrestha, A. et al. Generation and Validation of miR-142 Knock Out Mice. PLoS One 10, e0136913, https://doi.org/10.1371/journal.pone.0136913 (2015).
Article CAS PubMed PubMed Central Google Scholar
Zhu, H. et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat. Protoc. 5, 550–560, https://doi.org/10.1038/nprot.2009.238 (2010).
Article CAS PubMed Google Scholar
Wiemer, E. A., Wenzel, T., Deerinck, T. J., Ellisman, M. H. & Subramani, S. Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules. J. Cell Biol. 136, 71–80, https://doi.org/10.1083/jcb.136.1.71 (1997).
Article CAS PubMed PubMed Central Google Scholar
Edwards, S. R. & Wandless, T. J. The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain. J. Biol. Chem. 282, 13395–13401, https://doi.org/10.1074/jbc.M700498200 (2007).
Article CAS PubMed Google Scholar

Download references

Author information

Authors and Affiliations

Department of Anesthesiology, Kindai University Faculty of Medicine, Osaka, Japan
Kei Houri, Takatoshi Tsujimoto & Shinichi Nakao
Kindai University Life Science Research Institute, Kindai University, Osaka, Japan
Tatsufumi Mori
Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine, Kindai University Faculty of Medicine, Osaka, Japan
Yuta Onodera, Toshiyuki Takehara, Takeshi Teramura & Kanji Fukuda

Authors

Kei Houri
View author publications
You can also search for this author in PubMed Google Scholar
Tatsufumi Mori
View author publications
You can also search for this author in PubMed Google Scholar
Yuta Onodera
View author publications
You can also search for this author in PubMed Google Scholar
Takatoshi Tsujimoto
View author publications
You can also search for this author in PubMed Google Scholar
Toshiyuki Takehara
View author publications
You can also search for this author in PubMed Google Scholar
Shinichi Nakao
View author publications
You can also search for this author in PubMed Google Scholar
Takeshi Teramura
View author publications
You can also search for this author in PubMed Google Scholar
Kanji Fukuda
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

T. Teramura and K.F. were responsible for conceptualization. Y.O. and T. Teramura designed the studies. K.H., Y.O., T.M., T. Tsujimoto, T. Takehara and S.N. performed biological experiments. Y.O. and T. Takehara provided data analysis and interpretation. K.H. and T. Teramura. wrote the manuscript. All authors contributed to and approved the final manuscript.

Corresponding author

Correspondence to Takeshi Teramura.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplemental information.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Houri, K., Mori, T., Onodera, Y. et al. miR-142 induces accumulation of reactive oxygen species (ROS) by inhibiting pexophagy in aged bone marrow mesenchymal stem cells. Sci Rep 10, 3735 (2020). https://doi.org/10.1038/s41598-020-60346-2

Download citation

Received: 07 May 2019
Accepted: 06 February 2020
Published: 28 February 2020
DOI: https://doi.org/10.1038/s41598-020-60346-2

This article is cited by

MicroRNA expression and oxidative stress markers in pectoral muscle of broiler chickens fed diets supplemented with phytobiotics composition
- Karolina A. Chodkowska
- Marcin Barszcz
- Anna Tuśnio
Scientific Reports (2024)
Non-coding RNAs in photoaging-related mechanisms: a new paradigm in skin health
- Mohammad Hasan Soheilifar
- Nastaran Masoudi-Khoram
- Shima Ghorbanifar
Biogerontology (2022)
Integrated miRNA-mRNA transcriptomic analysis of hepatopancreas reveals molecular strategies in Chinese mitten crab (Eriocheir sinensis) under acute nitrite stress
- Longlong Fu
- Tianheng Gao
- Jianlin Pan
Aquaculture International (2021)
Protein quality control of cell stemness
- Pengze Yan
- Jie Ren
- Guang-Hui Liu
Cell Regeneration (2020)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.