We previously reported that hsa-miR-520d-5p is functionally involved in the induction of the epithelial–mesenchymal transition and stemness-mediated processes in normal cells and cancer cells, respectively. On the basis of the synergistic effect of p53 upregulation and demethylation induced by 520d-5p, the current study investigated the effect of this miRNA on apoptotic induction by ultraviolet B (UVB) light in normal human dermal fibroblast (NHDF) cells. 520d-5p was lentivirally transfected into NHDF cells either before or after a lethal dose of UVB irradiation (302 nm) to assess its preventive or therapeutic effects, respectively. The methylation level, gene expression, production of type I collagen and cell cycle distribution were estimated in UV-irradiated cells. NHDF cells transfected with 520d-5p prior to UVB irradiation had apoptotic characteristics, and the transfection exerted no preventive effects. However, transfection with 520d-5p into NHDF cells after UVB exposure resulted in the induction of reprogramming in damaged fibroblasts, the survival of CD105-positive cells, an extended cell lifespan and prevention of cellular damage or malfunction; these outcomes were similar to the effects observed in 520d-5p-transfected NHDF cells (520d/NHDF). The gene expression of c-Abl (Abelson murine leukemia viral oncogene homolog 1), ATR (ataxia telangiectasia and Rad3-related protein), and BRCA1 (breast cancer susceptibility gene I) in transfectants was transcriptionally upregulated in order. These mechanistic findings indicate that ATR-dependent DNA damage repair was activated under this stressor. In conclusion, 520d-5p exerted a therapeutic effect on cells damaged by UVB and restored them to a normal senescent state following functional restoration via survival of CD105-positive cells through c-Abl-ATR-BRCA1 pathway activation, p53 upregulation, and demethylation.
The effects of ultraviolet (UV) irradiation on human skin at the molecular and physiological level have been previously examined. UV irradiation induces cell cycle arrest and apoptosis by generating reactive oxygen species that can lead to oxidative base damage, such as 8-hydroxydeoxyguanosine and thymine glycol in DNA, causing single- or double-strand breaks.1,2 If not repaired in a timely manner, these UV-induced photolesions can cause severe structural distortions in DNA and affect important cellular processes, including DNA replication and transcription.3
miRNAs have been shown to regulate many biological processes such as embryonic development, cell differentiation, apoptosis, proliferation, pluripotency and biogenesis, and they are involved in a wide range of human diseases, including cancer.4,5 miRNAs protect the skin from damage due to brief UV irradiation and regulate miRNA function within miRNA networks.6 Well-known interactions between UV and miRNA function include the following: miR16 is involved in response to DNA damage;7 miR-155 and miR-101 are involved in the response to photoaging;8,9 miR-22, miR-125b and miR-19 are involved in cell survival;10,
We have reported that the human miRNA hsa-miR-520d-5p has the potential to convert undifferentiated or even well-differentiated cancer cells to benign or normal states in vivo via stemness-mediated processes23 and to revert human fibroblasts to mesenchymal stem cells.24 Because 520d-5p extends the lifespan of fibroblasts with this reprogramming mechanism and has never induced tumors in vivo, we presumed that this small molecule has anti-aging or aging-resistant effects against UV damage. In this limited scope, we focused on the gene expression in a small-cell population in response to lethal UVB irradiation and attempted to identify the role of hsa-miR-520d-5p in human fibroblasts, including the potential to salvage the damage caused by UVB. In addition, in this study, we attempted to determine the mechanism of cell survival in response to a lethal insult of UV radiation.
Observation of 520d/NHDF cells (transfectants) after UVB irradiation
All of the cells (520d/NHDF) lentivirally transfected with 520d-5p before UVB irradiation exhibited cell death without a preventive effect. However, some of the transfectants that received the miRNA after UVB irradiation survived (Figure 1a), showing an increased lifespan, unlike the parental cells (NHDF) or the scramble/NHDF cells. Because stemness induction by 520d-5p after UV irradiation of NHDF cells was reproduced between 2 h and 3 days after each irradiation, 520d-5p expression in NHDF cells transfected after irradiation (irradiation/transfection: a therapeutic trial) was estimated and confirmed on the 1st, 2nd and 35th day after transfection (Figure 1b). We could not quantify expression in the NHDF cells irradiated after transfection (transfection/UVB irradiation: a preventative trial) due to severe cellular damage.
Estimation of transfectant survival using flow cytometry
To evaluate the apoptotic cells after UVB irradiation, we performed flow cytometry on the fourth day after irradiation (the second day after transfection) using annexin-V. Only the 520d/NHDF cells transfected post-irradiation exhibited a reduced apoptotic effect, with 1.5-fold more cells advancing to S phase (Figure 2; third row from top) compared with the scramble/NHDF cells (Figure 2; second row from top) and 520d-5p/NHDF cells transfected pre-irradiation (Figure 2; bottom).
Transcriptional expression analysis in transfectants
Next, we examined gene expression in 520d/NHDF cells as previously reported.24 Although CAS3 was significantly downregulated (P<0.01 by Mann–Whitney U-test; n=4, data not shown), p53 was transiently upregulated, but its expression gradually decreased during the observation period. However, human telomerase reverse transcriptase (hTERT) expression was not affected by the transfection. CD105 expression was observed in cells a month after transfection and continued until cell death. Unexpectedly, ataxia telangiectasia mutated (ATM) was significantly downregulated throughout the course after irradiation (P<0.01 by Mann–Whitney U-test; n=4; Figure 3; see Supplementary Figure 1 for the detail statistical analyses).
Estimation of RNA methylation level in transfectants
Because RNA levels have been shown to be more reliable to estimate epigenetic changes in a hepatoma cell line (HLF) than DNA levels,24 we examined RNA methylation levels using NHDF cells in this study. The demethylation process was confirmed using total RNA m6A (%) in 520d/NHDF cells and NHDF cells subjected to irradiation/transfection. In the control NHDF cells, demethylation occurred at 5 weeks, and the methylation process was elevated during senescence. UVB irradiation and the subsequent transfection by 520d-5p induced significantly lower levels of RNA methylation (P<0.01 by Mann–Whitney U-test; n=4) similar to those observed in the 520d/NHDF cells (Figure 4a).
Estimation of functional restoration in transfectants
Collagen production from either control NHDF cells or NHDF cells treated with irradiation/transfection was evaluated. Compared with the NHDF cells, the cells transfected after irradiation had a lower level of collagen production except for the first spheroid populations, which showed significantly higher levels (P<0.01 by Mann–Whitney U-test; n=4). A subset of the senescent population retained higher levels of collagen production (Figure 4b). CD105 expression in the irradiated 520d/NHDF cells was confirmed up to 75 days after transfection (Figure 5a).
Estimation of differentiation potency in transfectants
Subsequently, we examined whether adipogenic, osteogenic, or chondrogenic differentiation was induced by differentiating agents in these senesced 520d/NHDF cells treated with irradiation (77 days after transfection). Ninety-one days after transfection (2 weeks after the stimulus for each differentiation), osteocalcin expression was induced, but neither FABP nor aggrecan were expressed (Figure 5b).
Insight into the mechanism of cell survival
A luciferase reporter assay revealed that one of the candidate targets of hsa-miR-520d-5p is ATM, and three algorithms predict that eight binding sites in the 3ʹ-UTR are targeted sites of 520d-5p (Figure 6a). Two of the eight target sites were confirmed as binding sites using hsa-miR-520d-5p and pMIRNA1/520d-5p (Figure 6b).
Although the transcriptional regulation and post-translational modification of proteins in response to UV irradiation has been well studied, the precise role of functional and regulatory molecules (including miRNAs) in the UV stress response of human fibroblasts has not been fully elucidated.25,
In the current study, NHDF cells without any treatment and scramble-treated NHDF (scramble/NHDF) cells died 3 days after UVB irradiation via apoptosis and not senescence.28,29 However, hsa-miR-520d-5p induced DNA-damaged fibroblasts to survive with reproducibility. The cells (520d/NHDF) that survived exhibited a lifespan that was extended 2.5 times, expressed a mesenchymal cell marker (CD105), and even differentiated towards an osteoblast-like status. The 520d/NHDF cells, in which RNA demethylation was induced, regained its ability to produce collagen.
To cope with the detrimental effects of UV exposure, the cellular machinery is driven by mounting a rapid, inducible, and transient response termed the ‘UV stress response.’ The first genetic response is the DNA damage repair system. After UV-induced damage, the expression of genes involved in DNA repair was silenced universally;30 conversely, many miRNAs were upregulated by UVB (280–320 nm) irradiation.31,32 In this study, OTUB1 (OTU deubiquitinase, ubiquitin aldehyde binding 1) was expressed following UVB irradiation, causing p53 stabilization, which was not downregulated even by lethal UVB irradiation33 (Figure 3). It may be very important in the DNA repair process that ‘ubiquitination and p53 upregulation’ precedes ‘c-Abl-mediated ATR-BCRA1 upregulation’ in the DNA repair process. However, the details regarding these mechanisms remain unclear.
Therefore, we examined transcriptional gene expression changes in irradiated 520d/NHDF cells due to the insufficient quantity of cells to analyze DNA levels (including telomere length) or translational levels. As we previously reported, this miRNA induces the upregulation of p53 and reprogramming via demethylation,23,24 and we presumed that general induction of miRNA upregulation after UV irradiation, especially 520d-5p upregulation, may repair damage even on lethal DNA strand(s) breaks. In transfectants (520d/NHDF), hsa-miR-520d-5p was expressed 5- to 10-fold higher compared with the parental cells (NHDF). In the DNA repair process, initial genetic activation was observed regarding c-Abl and ataxia telangiectasia and Rad3-related serine/threonine kinase (ATR), which were subsequently upregulated by 520d-5p 35 days after transfection,7,34,35 followed by the activation of BRCA1 (breast cancer 1, early onset).36,37 Six types of DNA damage repair (DDR) mechanisms are known as a modular structure of non-canonical DDRs, and mechanical stress induces ATR activation as an effector.38 Because ATM was continuously suppressed by 520d-5p, alternative ATR activation might drive towards a mechanical stress-based DDR pathway.38 It is thought that these pathways repair damaged DNA in an ATR-dependent manner under these conditions (Figure 7).
The 520d/NHDF cells that survived did so as a result of clonal expansion in the culture dish, and we examined whether they were regarded as normal cells without cancerous properties. These cells exhibited a prolonged but not infinite lifespan; they were positive for CD105 at the cell surface and in the cytoplasm; and they differentiated towards an osteoblastic lineage (Figure 5).24,39 Although the tumorigenicity of 520d/NHDF cells could not be examined due to senescence, we concluded that they escaped from the critical irradiation and senesced as normal cells because they did not exhibit immortalized properties.
UVB-induced DNA damage leads to the accumulation of mutations, predisposing people to carcinogenesis and premature aging. The genetic loss of nucleotide excision repair leads to severe disorders, including xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome, which are associated with a predisposition to skin cancer at a young age as well as developmental and neurological conditions.40,41 The regulation of nucleotide excision repair is an attractive avenue to either prevent or reverse these detrimental consequences.42 miRNAs may become a key to clarify the novel mechanism underlying the diseases because only a therapeutic effect was observed after inducing hsa-miR-520d-5p expression after DNA damage.
Thus, the results suggest that hsa-miR-520d-5p acts as a UVB protective agent with a therapeutic effect on fibroblasts by driving the ATR-dependent DNA repair system (non-canonical pathway) and maintaining p53 transcriptional stability via deubiquitination (an inhibition of DNA double-strand break (DBS)-induced chromatin ubiquitination), resulting in the reversion of UVB-irradiated cells to a mesenchymal status.43 Applications of this discovery in the anti-aging domain can be expected, including aftercare upon outdoor work or direct exposure to sunlight. This ribonucleic acid may be a highly useful molecule with therapeutic potential against fibroblast-related diseases or damage.
Materials and methods
To determine the in vitro effects of hsa-miR-520d-5p on UV-irradiated fibroblasts and to explore its protective application for anti-aging therapy, we used three cell lines and a lentiviral vector. NHDF cells were provided by TAKARA BIO (Tokyo, Japan). To examine the effect of hsa-miR-520d-5p on normal cells in vitro, we used NHDF cells cultured in fibroblast basal medium (FBM)-2 using the fibroblast growth medium chemically defined (FGM-CD) Bullet Kit (TAKARA BIO). In addition, the human mesangial cell line 293FT (Invitrogen Japan K.K., Tokyo, Japan) was used to produce the hsa-miR-520d-expressing lentivirus. The 293FT cells were cultured in Dulbecco’s modified Eagle’s medium (WAKO, Tokyo, Japan) supplemented with 10% fetal bovine serum, 0.1 mmol/l minimum essential medium (MEM) non-essential amino acids, 2 mmol/l l-glutamine, and 1% penicillin/streptomycin.
Lentiviral vector constructs
To examine the effects of hsa-miR-520d-5p overexpression on normal cells, we transfected pMIRNA1-miR-520d-5p/green fluorescent protein (GFP; 20 μg; System Biosciences, Mountain View, CA, USA), pMIRNA-scramble/GFP or the mock vector pCDH/lenti/GFP (20 μg) into 293FT cells. To harvest viral particles, the cells were centrifuged at 170,000×g (120 min, 4 °C). The viral pellets were collected, and the viral copy numbers were measured using a Lenti-XTM quantitative reverse transcriptase PCR (qRT-PCR) titration kit (Clontech, Mountain View, CA, USA). For NHDF-Ad infection, 1.0×107 copies of the lentivirus were used per 10 cm culture dish. The sequences for hsa-miR-520d-5p and the scrambled sequence were 5′- CUACAAAGGGAAGCCCUUUC-3′ and 5′-GAGUCCGCCTCUATAGACAA-3′, respectively.
Complete NHDF cell death was achieved by UVB treatment (302 nm) for 17 min (0.51 J/cm2) using the UV cross linker CL-1000 (Cosmo Bio, Tokyo, Japan). The irradiated cells were cultured in 10 cm2 dishes, and apoptotic induction and cellular survival until senescence were observed. Cells were irradiated with UVB 5 days after the 520d-5p transfection to investigate the preventive potential; the therapeutic potential was investigated by 520d-5p transfection between 2 h and 2 days after UVB irradiation.
Hsa-miR-520d-5p expression in NHDF cells or transfectants
The miRNA fraction was extracted from cultured cells using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). Mature miRNA (hsa-miR-520d-5p: 25 ng/μl) was quantified with the Mir-XTM miRNA qRT-PCR SYBR Green kit (TAKARA BIO) according to the manufacturer’s instructions.
Fluorescence detection in cells
To estimate the efficacy of infection by the hsa-miR-520d-5p-expressing lentiviral vector, GFP expression was detected with an OLYMPUS IX71 microscope using a TH4-100 power supply (Tokyo, Japan).
Gene expression analysis by RT-PCR
Total RNA was extracted from cultured cells with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). The gels were run under the same experimental conditions. The PCR and data collection analyses were performed with a BioFlux LineGene (Toyobo, Nagoya, Japan). To analyze the RT-PCR results, all of the data except those for hTERT were normalized to the β-actin internal control. hTERT expression was estimated by the copy number, and the U6 small nuclear RNA was used as an internal control. The total RNA (25 ng/μl) was reverse transcribed and amplified using the KAPA SYBR FAST One-Step qRT-PCR kit (NIPPON Genetics, Tokyo, Japan). RNA quantification was confirmed by high reproducibility sequencing. The primer sequences used for either mRNA or miRNA quantification are shown in Supplementary Table 1.
Cell cycle analysis by flow cytometry
The cells were categorized into four groups. NHDF cells and the scramble-transfectants that received 17 min UVB irradiation were used as controls. To examine the therapeutic effect, hsa-miR-520d-5p was lentivirally transfected into NHDF cells two days after a 17 min exposure to UVB. Four days after UVB irradiation, the DNA content in each mitotic phase was estimated by flow cytometry. To examine the preventive effect, hsa-miR-520d-5p was lentivirally transfected into NHDF cells 5 days prior to the 17 min exposure to UVB irradiation. Two days after UVB irradiation, the cells were examined via flow cytometry. Cell cycle analysis was conducted to confirm the DNA content in the 520d/NHDF cells. After pretreating each cell group with propidium iodide and using the Annexin V-biotin apoptosis detection kit (eBioscience; San Diego, CA, USA), the DNA content was analyzed with a flow cytometer (Epics Altra; Beckman Coulter, Brea, CA, USA). Approximately 20,000 cells were assessed 3 days after transfection of one of the pMIRNA1-miR-520d-5p/GFP clones using EXPO32 ADC Analysis software. For cell cycle analysis, a single-cell suspension was washed once with cold PBS. The cell pellet was disrupted by shaking the tube gently and was fixed with 3.7% formalin in ddH2O added dropwise. The cells were then incubated at least overnight at 20 °C. After fixation, the cells were washed twice with cold PBS to remove the ethanol, resuspended at 1×106 cells per ml in PBS containing 100 U/ml RNase A and incubated for 50 min at 37 °C.
Measurement of total RNA N6-methyladenosine (m6A) percentage
N6-methyladenosine levels were measured in NHDF cells, scramble/NHDF cells and 520d/NHDF cells (200 ng of RNA from each) using the EpiQuik m6A RNA Methylation Quantification kit (Colorimetric) according to the manufacturer’s instructions (EPIGENTEK, Farmingdale, NY, USA).
Because we could not obtain a sufficient number of cells for western blotting, immunohistochemical studies were performed with antibodies to detect a multilineage-differentiating stress enduring (muse) cell marker (anti-CD105) (Cell Signaling Technology, Boston, MA, USA), markers of differentiation (anti-osteocalcin, anti-aggrecan, or anti-fatty-acid-binding protein (FABP) antibodies) and mesenchymal stem cell markers according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). NHDF cells were infected with lentiviral particles that contained hsa-miR-520d-5p, and the transfectants were harvested and transferred either to a new culture dish for microscopic examination or onto a slide chamber for immunostaining.
Type I collagen quantification
To examine the ability of fibroblasts or 520d-transfected fibroblasts to produce type I collagen, 200 μl of supernatant was collected from cultures comprising 1×106 cells in 25 cm2 dishes, and the quantity of collagen produced by the 520d/NHDF cells was measured using the Human Collagen Type I ELISA kit (ACEL, Sagamihara, Japan).
Differentiation of 520d-NHDF cells by differentiation-inducing agents
Following treatment with differentiation agents, the induction of osteogenic, chondrogenic and adipogenic differentiation in the 520d/NHDF cells was examined using the Mesenchymal Stem Cell Identification kits (R&D Systems, Minneapolis, MN, USA).
Prediction of ATM as target genes of hsa-miR-520d-5p
The potential target genes for hsa-miR-520d-5p (MIMAT0002855: 5ʹ-CUACAAAGGGAAGCCCUUUC-3ʹ) were predicted using three databases (RNA22-HAS: http://cm.jefferson.edu/rna22v1.0, DIANA-microT: http://www.hsls.pitt.edu/obrc/index.php?page=URL1253808676, and microRNA.org: http://www.microrna.org/microrna/home.do). To investigate the binding of hsa-miR-520d-5p to the 3ʹ-UTR of ATM, sequences (or mismatch sequences) that corresponded with predictive hsa-miR-520d-5p binding sites were ligated into the multiple cloning site (MCS) (PmeI and XhoI) of the psiCHECK-2 plasmid (Promega KK, Tokyo, Japan). The signals were detected using the LightSwitch Luciferase Assay Reagent (Active Motif, Carlsbad, CA, USA). Synthesized hsa-miR-520d-5p (KOKEN, Tokyo, Japan) or the control vector pMIRNA1/GFP (System Biosciences, Mountain View, CA, USA) were co-transfected into NHDF cells together with each prepared luciferase expression vector. Synthesized hsa-miR-520d-3p (MBL, Nagoya, Japan) and pLKO.1 (Addgene, Cambridge, MA, USA) with mismatch sequences were used as controls. Forty-eight hours after transfection, luciferin expression (RLU) was measured with an Infinite F500 microplate reader (TECAN, Mannedorf, Switzerland). The RLU (Renilla) was standardized to a control reading (n=3). The following six pairs of sequences were inserted into psiCHECK-2: ATM-3ʹ-UTR sense 1, 5′-TCGAGCAATGTGTGTTCTTTGTATTT-3′; antisense 1, 5′-AAATACAAAGAACACACATTGC-3′; ATM-3ʹ-UTR sense 2, 5′-TCGAATTTTTTTTATTTTTTGTAGTTT-3′; ATM-3ʹ-UTR antisense 2, 5′-AAACTACAAAAAATAAAAAAAA-3′; ATM-3ʹ-UTR mismatch sense 1, 5′-TCGAGCAATGTGTGAACTTTGTATTT-3′; mismatch antisense 1, 5′-AAACTACAAAAAATAAAAAAAA-3′; ATM-3ʹ-UTR mismatch sense 2, 5′-TCGAATTTTTTTTATAATTTGTAGTTT-3′; and mismatch antisense 2, 5′-AAACTACAAAAAATAAAAAAAA-3′.
The Mann–Whitney U-test, t-test or one-way analysis of variance were used for comparisons between the control cells, the mock-treated cells and the hsa-miR-520d-5p-transfected cells results with one observed variable. P<0.05 was considered statistically significant (*P<0.05, **P<0.01). In the box plots, the top and bottom of each box represent the 25th and 75th percentiles, respectively, providing the interquartile range. The line through the box indicates the median, and the error bars indicate the 5th and 95th percentiles.
This work was supported by a Grant-in-Aid for Research for Promoting Technological Seeds B (development type), the Takeda Science Foundation, the Princess Takamatsu Cancer Research Fund (11–24313), the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (Exploratory Research) of the Japan Science and Technology Agency (JST) and the JSPS KAKENHI Grant (Grant-in-Aid for Challenging Exploratory Research) Number 23659285.