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.

研究背景：砷是一种广泛分布的天然有毒元素。长期经口摄入砷会引发多种恶性肿瘤，尤以皮肤癌最为常见。目前砷诱导癌变的具体分子机制尚未完全阐明，但多项研究证实，基因突变并非其驱动因素，而微小RNA（microRNA，miRNA）的表达异常在其中发挥了关键作用。慢性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，该细胞系常被用作砷诱导皮肤癌变的经典研究模型。研究假说：长期暴露于亚砷酸盐的HaCaT细胞中，微小RNA（miRNA）的早期表达变化可揭示细胞恶性转化的早期分子进程。实验方法：将HaCaT细胞分别在含0 nM与100 nM亚砷酸钠（NaAsO₂）的培养基中培养3周与7周。提取总RNA，采用Affymetrix基因芯片检测miRNA与信使RNA（messenger 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，并利用MetaCore™软件对靶基因进行通路注释与富集分析。采用蛋白质印迹（western blot）实验检测MDM2、HMGB1及TP53的mRNA与蛋白表达水平。实验结果：在培养3周与7周时，多个参与其他肿瘤系统癌变通路的miRNA与mRNA均出现显著差异表达。通过miRNA与mRNA表达调控网络分析，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位点的低乙酰化状态提示，暴露于亚砷酸盐的细胞中诱导产生的TP53并无转录激活活性。研究结论：本研究数据提供了充分证据，表明miRNA及其靶mRNA的早期表达变化可能参与了砷诱导的癌变过程。