Data from: Differentially expressed mRNA targets of differentially expressed miRNAs predict changes in the TP53 axis and carcinogenesis related pathways in human keratinocytes chronically exposed to arsenic

Background: Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Hypothesis: Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. Methods: HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite™ software, and mapped to their pathways using MetaCore™ software. MDM2, HMGB1 and TP53 mRNA and protein levels were assayed by western blot. Results: Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Conclusions: Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis.背景：砷是一种广泛分布的天然有毒元素。慢性砷摄入可引发多种癌症，尤以皮肤癌最为常见。目前砷诱导癌变的机制尚未完全阐明，但多项研究表明，突变并非其驱动因素，而微RNA（microRNA）表达改变发挥了重要作用。长期低浓度亚砷酸盐暴露可使永生化人角质形成细胞（HaCaT）发生恶性转化，该细胞系可作为砷诱导皮肤癌变的研究模型。 假说：慢性暴露于亚砷酸盐的HaCaT细胞中，微RNA（miRNA）表达的早期变化可揭示细胞恶性转化的早期进程。 方法：将HaCaT细胞分别在含0/100 nM亚砷酸钠（NaAsO2）的培养基中培养3周和7周。提取总RNA，采用Affymetrix微阵列芯片检测miRNA与信使RNA（mRNA）的表达水平。从TargetScan 6.2数据库获取差异表达miRNA的靶基因，通过Partek Genomic Suite™软件将其与差异表达mRNA取交集，并借助MetaCore™软件对靶基因进行通路注释。采用蛋白质印迹法（western blot）检测MDM2、HMGB1及TP53的mRNA与蛋白表达水平。 结果：在培养3周和7周时，已有大量参与其他系统癌变通路的miRNA与mRNA出现差异表达。通过miRNA与mRNA表达网络预测得到包含MDM2、HMGB1在内的TP53调控网络。总TP53蛋白及TP53-S15位点磷酸化水平均被诱导上调，但TP53-K382位点低乙酰化提示，在砷暴露细胞中诱导产生的TP53处于失活状态。 结论：本研究数据有力证明，miRNA及其靶mRNA的早期变化可能参与了砷诱导的癌变进程。