RNA-seq of UV irradiated human epidermal melanocytes from neonatal foreskin, HEMn-DP2.

UVR is the principal risk factor for melanoma development. While the role of UVR in DNA mutagenesis is incontrovertible, it remains controversial what role UVR-induced mutations play in melanoma genesis. To better understand how UVR is contributing to melanoma development, we investigated the non-mutational effect of UVR on the epigenome, specifically DNA methylation. Aberrant DNA methylation changes are a hallmark in melanoma; however, there are few reports on the effects of UVR on DNA methylation. We exposed melanocytes to UVR and cultured them for one-month to detect hertible and stable changes in DNA methylation. We found both hyper and hypo methylated sites after UVR exposure. While many of these changes occured outside of promoters and areas of active gene expression, there were changes in promoter DNA methylation changes that correlated with changes in gene expression. These changes also correlated with those found in melanoma and UVR sensitive sites were prognostic of patient overall survival. Our work shows for the first time UVR induced DNA methylation changes in melanocytes and may be another way in which UVR contributes to melanoma development. Overall design: Melanocyte cells were UV irradiated with a broad band UVR spectrum ( UVA & UVB) for a total dos of 175 J/m2. Cells were cultured for one month before performing reduced representation bisulfite sequencing (RRBS). Genomic DNA was digested by MspI restriction enzyme, which recgonizes the CCGG sequence, and then subjected to bisulfite converstion. Methylated cytosines within CpG sites are protected from sodium bisulfite conversion and remain unchanged. Unmethylated cyotosines undergo a reaction with sodium bisulfite resulting in a base change from cytosine to uracil. This base change allows for the detection of methylated versus unmethylated CpG sites through high throughput sequencing.

紫外线辐射（Ultraviolet Radiation，UVR）是黑色素瘤发生的首要危险因素。尽管UVR在DNA诱变中的作用已毋庸置疑，但其诱导的突变在黑色素瘤发生过程中所发挥的具体作用仍存在争议。为深入阐明UVR推动黑色素瘤发生的具体机制，我们探究了UVR对表观基因组，尤其是DNA甲基化的非突变效应。异常DNA甲基化改变是黑色素瘤的标志性特征之一，但目前针对UVR对DNA甲基化影响的研究报道仍较为匮乏。我们将黑素细胞暴露于UVR环境中并进行为期1个月的体外培养，以此检测DNA甲基化的可遗传且稳定的改变。我们观察到，UVR暴露后同时出现了高甲基化与低甲基化位点。尽管多数此类改变发生于启动子区域及活跃基因表达区域之外，但仍有部分启动子区域的DNA甲基化改变与基因表达变化存在相关性。此类改变还与黑色素瘤组织中检测到的改变存在关联，且UVR敏感位点可作为患者总生存期的预后标志物。本研究首次证实了UVR可诱导黑素细胞发生DNA甲基化改变，这或许是UVR促进黑色素瘤发生的另一重要途径。总体实验设计：将黑素细胞采用宽谱UVR（包含UVA与UVB）进行照射，总辐射剂量为175 J/m²。照射后的细胞经1个月体外培养后，开展简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing，RRBS）。具体流程为：首先使用识别CCGG序列的MspI限制性内切酶消化基因组DNA，随后进行亚硫酸氢盐转化。CpG位点内的甲基化胞嘧啶可抵御亚硫酸氢盐的转化作用，保持原有碱基序列不变；而非甲基化胞嘧啶则会与亚硫酸氢钠发生反应，由胞嘧啶转变为尿嘧啶。通过高通量测序即可精准区分并检测CpG位点的甲基化与非甲基化状态。