Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, 211166, Jiangsu, People's Republic of China; The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, 211166, Jiangsu, People's Republic of China.
Department of Orthopaedics, the First Clinic Medical College, Nanjing Medical University, Nanjing 210011, Jiangsu, People's Republic of China.
Biochim Biophys Acta Mol Basis Dis. 2017 Mar;1863(3):753-763. doi: 10.1016/j.bbadis.2016.12.018. Epub 2017 Jan 3.
Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in regulation of genes. The mechanism for regulation, however, is unknown. Here, to determine if, in human keratinocyte (HaCaT) cells, circular RNAs are involved in arsenite-induced acceleration of the cell cycle, a circRNA microarray was performed to analyze the variability of circRNAs in arsenite-treated HaCaT (As-HaCaT) cells and in arsenite-transformed (T-HaCaT) cells in comparison to control HaCaT cells. Among the circRNAs up-regulated in both As-HaCaT cells and T-HaCaT cells, hsa:circRNA_100284 (circ100284) had the greatest increase and was chosen for further research. The presence of circ100284 was confirmed in HaCaT cells. In these cells, arsenite induced increases of EZH2 and cyclin D1 and accelerated the cell cycle. MicroRNA (miR)-217 suppressed the expression of EZH2 was involved in regulation of the cell cycle. Further, in HaCaT cells exposed to arsenite, EZH2 regulated the cell cycle by binding to the promoter of CCND1, which codes for cyclin D1. Moreover, knockdown of circ100284 with siRNA inhibited the cell cycle acceleration induced by arsenite, but this inhibition was reversed by co-transfection with circ100284 siRNA and by a miR-217 inhibitor. Knockdown of circ100284 with siRNA or transfected with miR-217 mimic inhibited the capacity of T-HaCaT cells for colony formation, invasion, and migration, effects that were reversed by co-transfection with a miR-217 inhibitor or by epigenetic expression of EZH2. These results suggest that, in HaCaT cells, arsenite increases circ100284 levels, which act as a sponge for miR-217 and up-regulate the miR-217 target, EZH2, which, in turn, up-regulates cyclin D1and CDK4, and thus accelerates the cell cycle and leads to malignant transformation. Thus, circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis. This establishes a previously unknown mechanism between arsenite-induced acceleration of the cell cycle and carcinogenesis.
环状 RNA（circRNA）是一类来源于前体 mRNA 的非编码 RNA，参与基因调控。然而，其调控机制尚不清楚。在这里，为了确定环状 RNA 是否参与亚砷酸盐诱导的人角质形成细胞（HaCaT）细胞周期加速，我们进行了环状 RNA 微阵列分析，以比较砷处理的 HaCaT（As-HaCaT）细胞和砷转化的（T-HaCaT）细胞与对照 HaCaT 细胞中环状 RNA 的变异性。在 As-HaCaT 细胞和 T-HaCaT 细胞中上调的环状 RNA 中，hsa：circRNA_100284（circ100284）的上调幅度最大，因此选择其进行进一步研究。在 HaCaT 细胞中证实了 circ100284 的存在。在这些细胞中，亚砷酸盐诱导 EZH2 和细胞周期蛋白 D1 的增加，并加速细胞周期。miR-217 抑制 EZH2 的表达参与细胞周期的调节。此外，在暴露于亚砷酸盐的 HaCaT 细胞中，EZH2 通过与编码细胞周期蛋白 D1 的 CCND1 启动子结合来调节细胞周期。此外，用 siRNA 敲低 circ100284 可抑制亚砷酸盐诱导的细胞周期加速，但这一抑制可通过共转染 circ100284 siRNA 和 miR-217 抑制剂逆转。用 siRNA 敲低 circ100284 或转染 miR-217 模拟物可抑制 T-HaCaT 细胞的集落形成、侵袭和迁移能力，而共转染 miR-217 抑制剂或表观遗传表达 EZH2 可逆转这些作用。这些结果表明，在 HaCaT 细胞中，亚砷酸盐增加 circ100284 水平，其作为 miR-217 的海绵并上调 miR-217 靶标 EZH2，EZH2 又上调细胞周期蛋白 D1 和 CDK4，从而加速细胞周期并导致恶性转化。因此，circ100284 通过 miR-217 对 EZH2 的调节，参与人类角质形成细胞中砷酸盐加速的细胞周期致癌作用。这建立了砷酸盐诱导的细胞周期加速与致癌作用之间以前未知的机制。
