Project description:Dr. Liu's research group is interested in studying the expression and functions of galectin-3, -7 and -12, in particular the roles of these proteins in inflammation and neoplasm. Members of the galectin family are known to participate in cellular homeostasis by modulating cell growth, controlling cell cycle progression, and inducing or inhibiting apoptosis. It is known that some galectins have similar functions. However, it is not fully understood whether they work cooperatively or not. As the outermost barrier of the body, skin is directly and frequently exposed to a prooxidative environment, including solar ultraviolet A (UVA), ultraviolet B (UVB) radiation, and air pollution. Several reports have shown that exposure of cells to UV increase or decrease the levels of galectins. For example, the amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of keratinocytes (Proc. Natl. Acad. Sci. USA 1999; 96:11329-34). Heat shock and subculturing decrease, while alkylating agents and UV-light increase galectin-3 (Cell Physiol Biochem 2000; 10:149-58). To analyze the change of all galectin gene expression profiles after UVB irradiation and to determine the presence or absence of coordinate regulation, we analyzed the gene expression profiles of keratinocytes exposed to UVB. Normal human epidermal keratinocytes (NHEK) were irradiated with 200 J/m2 of UVB. Total RNA will be extracted at 0, 6, 12 and 24 h after irradiation (duplicate) for analysis on the Glyco gene chip. Several reports have shown that exposure to UV light can regulate levels of galectin in skin. This study seeks to analyze the changes in all galectin gene expression profiles post-UVB irradiation to determine the presence or absence of coordinate regulation. In this study, normal human keratinocytes were irradiated with 200J/m2 of UVB. Total RNA was extracted at 0, 6, 12, and 24-hour post irradiation time points, in duplicate. Samples were hybridized and analyzed using the GLYCOv2 array.

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.

Dataset Information

UVB irradiation does not directly induce detectable changes of DNA methylation in human keratinocytes

Publications

Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells.

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