Dr Jie Zhang, Radiation Medicine Department of Institute of Preventive Medicine, Fourth Military Medical University, 169 Changle Xi Road, Xi'an, Shaanxi 710032, P.R. China
The skin is the largest outermost organ of the human body. It is vulnerable to various damages, such as ionizing radiation. Exploration of proliferation, senescence and radiosensitivity of skin cells contributes to the development of medical and cosmetic countermeasures against skin aging and toward injury protection. Human antigen R (HuR) is one of the most widely studied RNA-binding proteins and serves an important role in stabilization of mRNA and regulation of the expression of the target genes. To investigate the role of HuR in modulating proliferation, senescence and radiosensitivity of skin cells, the present study performed an
Regulation of gene expression is a complex process that induces gene expression in the cells providing spatiotemporal response to changes in environmental conditions, which serve as the molecular basis of cellular differentiation, morphogenesis and ontogeny in the organism (
Human antigen R (HuR; also known as HuA or ELAVL1) is one of the most extensively studied RBPs and serves an important role in the stabilization of mRNAs containing AU-rich elements (AREs) and regulation of the expression of the target genes (
The skin is the largest outermost organ of the human body. The primary role of the skin is to serve as a physical barrier, protecting our bodies from potential assault by foreign organisms or toxic substances. HuR is known to be modulated by mitogenic and stress-causing agents, including UV radiation (
In the present study, the third generation of four-plasmid lentivirus vector system was used. The lentiviral vector system was composed of four plasmids: The expression plasmid and three packaging vectors, including 1.5 µg pMDLg-pRRE, 1.5 µg pMD2.G and 1.5 µg pRSV-Rev Virus packaging helper plasmids. Lentiviral vectors and packaging vectors were transfected into 293T cells (Wuhan GeneCreate Biological Engineering Co., Ltd.). 293T cells were seeded with DMEM (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; cat. no. 04-001-1A; Biological Industries) and cultured in a 37˚C incubator with a 6-well plate of 2 ml/well. When the cell density reached 70-80%, it was used for transfection. The cells were cultured in serum-free medium before transfection. 2 µg expression plasmid, 1.5 µg PMDLG-PRRE, 1.5 µg PMD2.g and 1.5 µg PRSV-Rev virus packing assistant plasmid were diluted in 500 µl serum-free medium. Lipofectamine® 2000 (15 µl; Invitrogen; Thermo Fisher Scientific, Inc.) was diluted with 500 µl serum-free medium. After standing for 5 min, The DNA solution was mixed with Lipofectamine® 2000 solution and stood for 20 min at room temperature. Serum-free medium (1 ml) was taken from the 6-well plate and 1 ml plasmid and Lipofectamine® 2000 were dropped into the 6-well plate at 37˚C and 5% CO2 for 8 h. Following transfection, the culture medium was exchanged with DMEM (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; cat. no. 04-001-1A; Biological Industries). After 48 h, the supernatant containing the retroviral particles was collected and then concentrated by centrifugation at 4,000 x g for 10 min at 4˚C. The cell supernatant was filtered by a 0.45 µm filter into a 50 ml ultrafast centrifuge tube and 5X PEG8000 was added to precipitate at 4˚C overnight. After centrifugation at 7,000 x g, the supernatant was discarded and the precipitation was redissolved with 10 ml PBS. The viral supernatants were added to the upper layer of 20% sucrose solution, centrifuged at 20,000 x g for 2 h and 4˚C. The precipitate was suspended with 1 ml PBS and filtered by a 0.22 µm filter for sterilization. The virus suspension was separated into 50 µl and stored at -80˚C. A total of 1x105 cells/well were transduced with viral supernatants. The cells were collected for subsequent experiments after being cultured in fresh medium for 48 h at 37˚C.
The human HuR coding region was amplified by polymerase chain reaction (PCR) using a primer pair specific to HuR. The fragments were inserted into the lentiviral expression vectors (LV-HuR) and then packaged into viral particles. The negative control lentiviral (LV-NC) was constructed by not inserting any sequences. Lentivirus silencing HuR through shRNAs were obtained from Hanbio Biotechnology (containing RFP). The targeting sequences of shRNA control (sh-NC) and four shRNA targeting HuR (sh-HuR-1, sh-HuR-2, sh-HuR-3 and sh-HuR-4) are listed in
Human keratinocyte HaCaT (cat. no. iCell-h006, iCell Bioscience Inc.) and human skin fibroblast WS1 (cat. no. CRL-1502T; ATCC) cells were maintained in DMEM supplemented with 10% FBS and 100 U/ml penicillin-streptomycin at 37˚C and a 5% CO2 atmosphere. For transfection, cells were transfected by Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) with viral particles. Cells were exposed to a single dose (20 Gy) of X-rays using the linear accelerator (RadSource) at a dose rate of 1.15 Gy/min. The HaCaT cell line was obtained from iCell Bioscience Inc. DNA was extracted with Axygen genome extraction kit (cat. no. AP-GX-250; Axygen® AxyPrep DNA Gel Extraction Kit; Corning, Inc.) and amplified with 21-str amplification scheme. STR loci and sex gene Amelogenin were detected on an ABI 3730XL genetic analyzer (Applied Biosytems; Thermo Fisher Scientific, Inc.). The results showed that the cell line was completely matched by DNA typing in cell line retrieval and the cell name was HACAT and the cell number was 771 according to DSMZ database. No multiple alleles were found in this cell line.
After cells reached 90% confluence, total RNA was extracted using TRIzol® reagent (cat. no. 15596018; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. RNA was reverse transcribed using a reverse transcription kit (cat. no. K1691; RevertAid RT Reverse Transcription kit; Thermo Fisher Scientific, Inc.) under 42˚C for 60 min and 70˚C for 5 min. The mRNA level of
The cells were lysed in lysis buffer (Promega Corporation) and centrifuged at 4˚C, 12,000 x g for 10 min. The supernatant was collected and subjected to western blotting. Protein concentration was subsequently measured using a BCA Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Protein (50 µg) from each lysate was fractionated by 10% SDS-PAGE. The samples were electrophoresed for 2 h and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). After being blocked with 5% BSA in TBS-0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were blotted with HuR (Abcam; cat. no. ab220342) or α-Tubulin (Beyotime Institute of Biotechnology; cat. no. AF0001) primary antibodies at 1:1,000 dilutions. The membranes were then incubated with the appropriate horseradish peroxidase-coupled secondary antibody (Beyotime Institute of Biotechnology; cat. no. A0277) at a 1:2,000 dilution for 1 h at room temperature. After the membranes were washed with TBST, the blots were incubated with enhanced chemiluminescence (ECL) stable peroxide solution (Beyotime Institute of Biotechnology). All blots were visualized using a FluoroChem MI imaging system (Alpha Innotech Corporation) at room temperature. Densitometry was performed using ImageJ v1.8.0-172 software (National Institutes of Health).
Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.) assay. HaCaT and WS1 cells were plated in 96-well plates and cultured for 12 h at 37˚C in a CO2 incubator. The cells were transfected with LV-NC, LV-HuR, sh-NC, sh-HuR-1 and sh-HuR-2 viral particles. After treatment for 24 and 48 h at 37˚C in a CO2 incubator, the cells were then incubated with 10 µl CCK-8 for 4 h. Then the optical density (OD) at 450 nm was measured using a Microplate Reader (Bio-Rad Laboratories, Inc.). The viability index was calculated as experimental OD value/control OD value. Three independent experiments were performed in quadruplicate.
The proliferation of HaCaT and WS1 cells transfected with different viral particles was determined by 5-Ethynyl-2'-deoxyuridine (EdU) assay kit. Cells were pre-infected with LV-NC, LV-HuR, sh-NC, sh-HuR-1 and sh-HuR-2 viral particles for 24 h at 37˚C in a CO2 incubator before receiving sham or 20 Gy X-ray irradiation, after irradiation for 48 h at 37˚C in a CO2 incubator, cells were labeled with 50 µM EdU (Guangzhou RiboBio Co., Ltd.) for 4 h at 37˚C in a CO2 incubator. Then, the cells were fixed with 4% formaldehyde for 15 min at room temperature and treated with 0.5% Triton X-100 for 20 min at room temperature. The cells were washed with PBS for three times and treated with 100 µl of 1X ApolloR (EdU; Guangzhou RiboBio Co., Ltd.) reaction cocktail in the dark at room temperature for 30 min. Subsequently, the DNA of each well of cells were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; MilliporeSigma) for 30 min at room temperature and observed under a fluorescence microscope (Olympus Corporation).
For standard clonogenic assays, cells were re-suspended and seeded into six-well plates at 200 cells/well, cells were pre-infected with LV-NC, LV-HuR, sh-NC, sh-HuR-1 and sh-HuR-2 viral particles 24 h at 37˚C in a CO2 incubator before receiving sham or 20 Gy X-ray irradiation. The cells were grown from 7-10 days to allow for colony formation and were subsequently fixed and stained using crystal violet. Colonies consisting of >50 cells were counted as a clone.
Apoptosis was measured using propidium iodide (PI)/Annexin-V double staining following manufacturer's instructions (BD Biosciences). Cells were pre-infected with LV-NC, LV-HuR, sh-NC, sh-HuR-1 and sh-HuR-2 viral particles 24 h at 37˚C in a CO2 incubator before receiving sham or 20 Gy X-ray irradiation. After irradiation for 48 h, HaCaT and WS1 cells were harvested and apoptotic fractions were measured using flow cytometry (Beckman Coulter, Inc.). The Annexin-V+/PI- cells are early in the apoptotic process, the Annexin-V+/PI+ cells indicating late apoptosis. The percentage of both types of cells was counted. To compute the percentage of apoptotic cells, a flow cytometer (FACSCalibur; BD Biosciences) with ModFit's LT v.3.0 software (BD Diagnostics) was used for data analysis.
The cell senescence of HaCaT and WS1 cells transfected with different viral particles was determined by β-galactosidase staining. After the cells were transfected with LV-NC, LV-HuR, sh-NC, sh-HuR-1 and sh-HuR-2 viral particles for 24 h at 37˚C in a CO2 incubator. The cell senescence staining was performed according to the β-galactosidase staining kit (Beyotime Institute of Biotechnology).
HaCaT and WS1 cells were fixed in 2% formaldehyde/0.2% glutaraldehyde for 5 min at room temperature. β-Galactosidase staining solution containing X-gal (cat. no. C0602; Beyotime Institute of Biotechnology) was added after rinsing with PBS. The cells were then incubated for 6-10 h in a 37˚C incubator without CO2. Senescent cells (stained blue) were observed and images were captured using light microscopy (Olympus Corporation; magnification, x10) and positive staining areas were calculated by determining the percentage of SA-β-gal+ cells in five random fields in each of the three wells.
ROS levels after irradiation were determined using the ROS assay kit (Beyotime Institute of Biotechnology). HaCaT and WS1 cells were pre-infected with sh-NC, sh-HuR-1 and sh-HuR-2 viral particles 24 h at 37˚C in a CO2 incubator before receiving 20 Gy X-ray irradiation. After irradiation for 24 h, cells were labeled with 10 µM 2,7-dichlorofluorescein diacetate (DCFH-DA) at 37˚C for 30 min. Then, the cells were fixed with 4% formaldehyde for 15 min at room temperature and washed with PBS for three times, after then treated with 5 µg/ml Hoechst 33258 (Beyotime Institute of Biotechnology) for 30 min and observed under a fluorescence microscope (PerkinElmer, Inc.).
Total RNA was extracted from HaCaT cells infected with LV-NC, LV-HuR, sh-NC or sh-HuR-1 lentiviruses (n=3) using TRIzol® reagent (Thermo Fisher Scientific, Inc.). For RNA high-throughput sequencing, RNA libraries were created from each group using the NEBNext Ultra Directional RNA Library preparation kit from Illumina, Inc. The main steps in the workflow involved the removal of ribosomal RNA, the fragmentation of total RNA, reverse transcription and second-strand complementary DNA (cDNA) synthesis, end repair, dA tailing and adaptor ligation. The products of these reactions were purified and enriched by polymerase chain reaction to create the final cDNA library. The libraries were then sequenced using an Illumina HiSeq2500 (paired-end sequencing; Illumina, Inc.).
The RNA sequence row data was obtained by RNA-seq on the Illumina platform. Then, the reads were clipped and trimmed to avoid low-quality data using Trim Galore (
For the analysis of differentially expressed genes, the clean data for each sample were aligned to the rat reference genome (ftp://ftp.ensembl.org/pub/release-83/fasta/rattus_norvegicus/dna/) using TopHat (version 2.0.10) software (
The database for annotation, visualization and integrated discovery (DAVID;
RIP was performed as described previously (
The data were evaluated using either unpaired two-sided Student's t-tests or one-way analysis of variance to determine statistical significance after confirming that the data met appropriate assumptions. For all experiments, three biological replicates were analyzed for each condition and presented as the mean ± standard error of the mean. Differences between two groups were determined using a paired Student's t-test and differences among >2 groups were analyzed by one-way analysis of variance and Tukey post hoc tests. Statistical analysis was performed using Prism 7 software (GraphPad Software, Inc.). Data are expressed as mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.
To test the overexpression of LV-HuR, the present study measured HuR expression by RT-qPCR and western blotting. The results showed that the expression of HuR mRNA in LV-HuR cells was significantly higher compared with that in LV-NC cells (
Initially, the present study investigated the effect of HuR on the proliferation of skin cells by a CCK-8 assay. The results showed that overexpression of HuR promoted the growth of HaCaT and WS1 cells (
Temporal and environmental aging influences skin structure and functions, including the skin barrier and elastic and mechanical properties of cutaneous tissue associated with alterations of biomechanical properties of skin cells (
Since HuR influences the stability of mRNAs (
Radiation-induced skin injury is a common complication after radiation accidents, tumor radiation therapy and bone marrow transplantation pretreatment (
The present study used RIP combined with RNA-Seq to detect changes in the HuR-binding sequence and expression profile of WS1 cells after 0 or 5 Gy irradiation (
HuR is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs and post-transcriptionally regulates the fate of target RNAs. HuR regulates cellular responses to differentiation, senescence, inflammatory factors and immune stimuli by tightly controlling the post-transcriptional fate of specific mRNAs (
HuR has been shown to associate with numerous transcripts, including coding and noncoding transcripts, and controls their splicing, localization, stability and translation (
Skin aging is a slow and complex process subjected to intrinsic alterations at the cellular, molecular and genetic levels and by exposure to extrinsic factors (
The results of RIP-Seq analyses identified 14 mRNAs that preferentially interacted with HuR after ionizing radiation. A number of these genes are known to participate in certain key cellular processes, including proliferation, apoptosis, immune response and metastasis. For example, ENDOCAN is a novel human endothelial cell-specific molecule mainly expressed in endothelial cells in various tissues (
In order for HuR to exert its effects, it must dimerize prior to binding its targets. Thus, targeting of HuR may offer the ability to modulate a broad range of HuR-mediated effects, by interfering with the actions of a single target. HUR served a positive role in modulating proliferation, senescence and radiosensitivity of skin cells and modulated downstream mRNAs implicated in multiple pathways in skin cells, providing a new therapeutic strategy for cosmetic treatments and to combat skin injury.
Not applicable.
The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository,
DY and SZ confirm the authenticity of all the raw data. DY and SZ conceived and designed the study. DY, KF and ZJ carried out the molecular biology studies. DY, YF and TY drafted the manuscript and figures, prepared the samples for RNA-seq and carried out the senescence studies. SZ, YS and JZ performed the statistical analysis. DY and SZ edited the manuscript. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
The overexpression and silencing of HuR in skin cells. Reverse transcription-quantitative PCR analysis of (A) the overexpression efficiency of lentiviral vectors and (B) the silencing efficiency of four shRNA targeting HuR gene (shRNA-HuR). *P<0.05, **P<0.01. HuR, Human antigen R; sh, short hairpin.
Overexpression and silencing of HuR affected the cell proliferation of HaCaT and WS1 cells. The cell viability of (A) overexpression and (B) silencing of HuR on HaCaT and WS1 was measured by CCK-8 assay. EdU-based staining of HaCaT and WS1 after (C) overexpression and (D) silencing of HuR. Scale bar, 100 µm. *P<0.05, **P<0.01. HuR, Human antigen R; sh, short hairpin; NC, negative control; LV, lentiviral.
Overexpression and silencing of HuR modulates the cell clonogenic survival and apoptosis of HaCaT and WS1. Clonogenic survival staining of HaCaT and WS1 after (A) overexpression and (B) silencing of HuR. Flow cytometry measured cell apoptosis of HaCaT and WS1 after (C) overexpression and (D) silencing of HuR. *P<0.05, **P<0.01. HuR, Human antigen R; sh, short hairpin; NC, negative control; LV, lentiviral.
Overexpression and silencing of HuR modulates the cell senescence of skin cells. (A) β-galactosidase staining of HaCaT and WS1 cells after overexpression of HuR. (B) β-galactosidase staining of HaCaT and WS1 cells after silencing of HuR. Representative images of staining and calculated positive cell percentage are shown. Scale bar, 100 µm. **P<0.01. HuR, Human antigen R; sh, short hairpin; NC, negative control; LV, lentiviral.
The effect of HuR on downstream mRNAs by RNA-Seq. (A) The heatmap of significant differently expressed mRNA (LV-HuR vs. LV-NC group). (B) Volcano plot of the significant differently expressed mRNA (LV-HuR vs. LV-NC group). (C) The heatmap of significant differently expressed mRNA (sh-HuR vs. sh-NC group). (D) Volcano plot of the significant differently expressed mRNA (sh-HuR vs. sh-NC group). HuR, Human antigen R; sh, short hairpin; NC, negative control; LV, lentiviral.
Overexpression and silencing of HuR affected the cell radiosensitivity of skin cells. (A) The ROS of HaCaT cells pre-infected with sh-NC, sh-HuR-1 or sh-HuR-2 viral after receiving 20 Gy X-ray irradiation. Scale bar, 20 µm. EdU staining of HaCaT and WS1 after (B) overexpression and (C) silencing of HuR with 20 Gy X-ray irradiation. Scale bar, 100 µm. Clonogenic survival staining of HaCaT and WS1 after (D) overexpression and (E) silencing of HuR with 20 Gy X-ray irradiation. Flow Cytometry measured cell apoptosis of HaCaT and WS1 after (F) overexpression and (G) silencing of HuR with 20 Gy X-ray irradiation. *P<0.05, **P<0.01. HuR, Human antigen R; ROS, reactive oxygen species; sh, short hairpin; NC, negative control; LV, lentiviral.
RIP-seq to screen HuR-interacted mRNAs upon radiation. (A) Experimental design of the HuR RIP-seq analysis. (B) Differentially gene regions in human chromosomes. Red and green bars represent upregulated and downregulated sites, respectively. (C) Percentage of the location of gene regions. (D) Heatmap of HuR RIP genes and corresponding mRNA after 0 or 5 Gy X-ray irradiation. RIP-seq, RNA immunoprecipitation and sequencing; HuR, Human antigen R; ROS, reactive oxygen species; sh, short hairpin; NC, negative control; LV, lentiviral.