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UVB drives different stages of epigenome alterations during progression of skin cancer [Bisulfite-seq]

ABSTRACT: Exposure to UVB irradiation results in multitude of cellular responses including generation of reactive oxygen species (ROS) and DNA damage and is responsible for non-melanoma skin cancers (NMSCs). Although genetic mutation is well documented, the epi-mutation, the alteration in epigenetics, remains elusive. DNA methylation is a stable and heritable epigenetic change in the control of gene expression. Furthermore, it is unclear how the epigenomic DNA methylome would change during progression of UVB-induced carcinogenesis. In this study, we utilized CpG Methyl-seq to identify a genome-wide DNA CpG methylation, to profile the DNA methylation in ultraviolet B (UVB)-irradiated SKH-1 mouse skin epidermis and non-melanoma skin papillomas at various stages. Agilent mouse SureSelect Methyl-seq Kit was used for enrichment of about 3.3 million CpG sites in the mouse genome. Simultaneously, RNA-seq was performed to examine the corresponding transcriptome alterations. Bioinformatic analysis was utilized to identify differentially methylated and differentially expressed genes. The methylation profiles in mouse epidermis were altered by UVB-irradiation as time progresses. Comparing all epidermis samples at week-15 versus week-25, UVB had great impact on DNA methylome alteration. Examination of the differentially methylated regions (DMRs), most of the DMRs were located in the distal intragenic (>3 kb upstream of TSS or downstream of 3’ UTR) and the promoter regions. Interestingly, at week-25 the methylation profiles between the whole skin versus the tumor samples were different. Ingenuity Pathways Analysis (IPA) identified many cancer related pathways including PTEN, p53, Nrf2 and inflammatory signaling in UVB-irradiation induced carcinogenesis. Cell cycle regulation and cell growth related pathways including State3 signaling and estrogen-mediated S-phase entry were also identified. Additionally, some novel genes involved in skin carcinogenesis that were not previously reported were differentially methylated, including Enf2, Mgst2, Vegfa, and Cdk4. The methylation and expression of these genes were validated by pyrosequencing and qPCR. Taken together, the current study provides novel profiles of methylation and transcriptomic changes at different stages of carcinogenesis in UVB-irradiation induced NMSC and offer potential targets for prevention and treatment of NMSC at different stages of human skin cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE122226 | GEO | 2019/03/20

REPOSITORIES: GEO

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Project description:Exposure to UVB irradiation results in multitude of cellular responses including generation of reactive oxygen species (ROS) and DNA damage and is responsible for non-melanoma skin cancers (NMSCs). Although genetic mutation is well documented, the epi-mutation, the alteration in epigenetics, remains elusive. DNA methylation is a stable and heritable epigenetic change in the control of gene expression. Furthermore, it is unclear how the epigenomic DNA methylome would change during progression of UVB-induced carcinogenesis. In this study, we utilized CpG Methyl-seq to identify a genome-wide DNA CpG methylation, to profile the DNA methylation in ultraviolet B (UVB)-irradiated SKH-1 mouse skin epidermis and non-melanoma skin papillomas at various stages. Agilent mouse SureSelect Methyl-seq Kit was used for enrichment of about 3.3 million CpG sites in the mouse genome. Simultaneously, RNA-seq was performed to examine the corresponding transcriptome alterations. Bioinformatic analysis was utilized to identify differentially methylated and differentially expressed genes. The methylation profiles in mouse epidermis were altered by UVB-irradiation as time progresses. Comparing all epidermis samples at week-15 versus week-25, UVB had great impact on DNA methylome alteration. Examination of the differentially methylated regions (DMRs), most of the DMRs were located in the distal intragenic (>3 kb upstream of TSS or downstream of 3’ UTR) and the promoter regions. Interestingly, at week-25 the methylation profiles between the whole skin versus the tumor samples were different. Ingenuity Pathways Analysis (IPA) identified many cancer related pathways including PTEN, p53, Nrf2 and inflammatory signaling in UVB-irradiation induced carcinogenesis. Cell cycle regulation and cell growth related pathways including State3 signaling and estrogen-mediated S-phase entry were also identified. Additionally, some novel genes involved in skin carcinogenesis that were not previously reported were differentially methylated, including Enf2, Mgst2, Vegfa, and Cdk4. The methylation and expression of these genes were validated by pyrosequencing and qPCR. Taken together, the current study provides novel profiles of methylation and transcriptomic changes at different stages of carcinogenesis in UVB-irradiation induced NMSC and offer potential targets for prevention and treatment of NMSC at different stages of human skin cancer.

Project description:Renin-angiotensin system regulates cell proliferation, differentiation, apoptosis and tissue remodeling in skin. Previouly, we reported that angiotensin-converting enzyme (ACE) expression and angiotensin II level were enhanced by repeated UVB-irradiation of hairless mice skin, and ACE inhibitor enalapril maleate accelerated recovery of UVB-induced wrinkle. In present study, we analyzed gene expression with DNA microarray and protein distribution with immunofluorecence method to clarify the process of wrinkle repairing with enalapril maleate in UVB-irradiated hairless mouse. In microarray analysis, we detected up-regulation of various extracullular matrix (ECM) genes and ECM related genes. In immunofluorescence, we confirm that type 1 collagen, fibrillin1, elastin, and dystroglycan 1 were decreased by repeated UVB-irradiation and the distribution of these components were improved by enalapril maleate treatment. In addition, ADAMTS2 and MMP-14, these were perticipate in ECM structure formation and degradation, were increased in enalapril maleate-treated mouse skin. While the functions of these molecules in skin is not clear yet, it is suggested that these molecules participate in improvement of wrinkles induced by UVB-irradiation. Male hairless mice of the SKH-1 strain were purchased from Charles River Laboratories Japan, Inc. (Tokyo, Japan). These animals were approximately 6 weeks old at the start of the experiment. They were fed a commercial diet (CRF-1, Oriental Yeast Co., Ltd, Tokyo, Japan) ad libitum and allowed free access to water. The dorsal region of the mouse was repeatedly irradiated with UVB for 10 weeks. Enalapril maleate treatment was started at 1 week after 10-week irradiation. One hundred microliters of 1% w/v enalapril maleate dissolved in 30 vol % ethanol solution or 30 vol % ethanol (control) was applied 5 times a week for 2 consecutive weeks to the whole dorsal skin of each of the mice. All experimental procedures using mice were approved by the Animal Experiment Committee of Tokyo University of Agriculture and Technology (approval number 24-82). The dosal region of the mouse was repeatedly irradiated with UVB for 10 weeks. Enalapril maleate treatment was started at 1 week after 10-week irradiation. One hundred microliters of 1% w/v enalapril maleate dissolved in 30 vol % ethanol solution or 30 vol % ethanol (control) was applied 5 times a week for 2 or 6 consecutive weeks to the whole dorsal skin of each of the mice. Each of the experimental groups comprised 6 mice. After 2- or 6-week drug treatment, skin samples were collected for microarray analysis.

Project description:UVA1 makes up ~75% terrestrial ultraviolet radiation (UVR) is increasingly being used for the treatment for inflammatory skin disorders. It is also the major UVR spectral source in tanning lamps, however we lack human data to understand its effects on gene expression and further consequences. Using erythemally equivalent doses of UVA1 (50J/cm2) and UVB (30mJ/cm2) ~1MED (minimal erythema dose), this study examines time dependent whole genome expression, mRNA and protein changes in 12 skin types I/II individuals. The top upregulated pathway at 6h is inflammation through IL17 signaling for UVA1 (p=1.16e-6) and UVB (p=2.1e-4). At 24h the top upregulated pathway is extracellular matrix remodeling for UVA1 (p=5.5e-7) and UVB (p=2.9e-22). We show key spectral differences with matrix metalloproteinases (MMP): UVB upregulates MMP1 mRNA (p=0.0062), MMP3 mRNA (p=0.0016), MMP10 mRNA (p=0.028) at 24h compared to UVA1, MMP12 mRNA is upregulated by UVA1 at 6h and not by UVB (p=0.02). In a further 3 skin type I/II we show that MMP12 protein is formed by UVA1 (p=0.04) at 24h and that DQM-bM-^DM-" collagen type IV(likely MMP12 activity) hydrolysis occurs by UVA1 (p=0.027) at 10h. DQM-bM-^DM-" collagen type I hydrolysis occurs predominantly by UVB at 24h (p=0.031). Both UVA1 and UVB upregulate MMP1 at 10h and 24h. There is more MMP1 and MMP12 protein and activity in the epidermis than in dermis in all volunteers. We conclude that the biological processes that occur after 24h in human skin in vivo in response to erythemally equivalent doses of UVA1 and UVB are similar however there are key spectral differences: UVB plays a larger role in upregulating key MMPs in the epidermis. MMP12 is a UVA1 specific biomarker found predominantly in the epidermis and maybe useful as a marker of UVA1 exposure. We hypothesise that its formation by UVA1 could be related to the ability of UVA1 to produce significantly more oxidatively generated DNA than UVB (8oxodG). 47 samples were processed. The RNA was isolated from human skin samples of individual donors pre- or post irradiation with UVA or UVB. Skin biopsies were taken either before, 6h post, or 24h post exposure. Different doses of UVA were applied. Each condition was available from five or nine individuals, as indicated in the samples description below.

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UVB drives different stages of epigenome alterations during progression of skin cancer [Bisulfite-seq]

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