无机砷通过抑制DHX9增加circRNA表达的机制研究

Circular RNAs are characterized by covalently closed loop structures with neither 5’ to 3’ polarity nor polyadenylated tail. Altered circRNAs expression has been reported in several diseases like cancer, heart disease, neurological disorders, diabetes and atherosclerosis. Our previous studies have shown that sodium arsenite had increased expression of circNUP107, circAKR1A1 and circDKC1, and decreased expression of linear NUP107 and linear AKR1A1 after treatment for 5 days. We observed a similar result that decitabine had increased expression of circNUP107, circAKR1A1 and circDKC1, and decreased expression of linear NUP107 and linear AKR1A1. Monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) did not changed RNAs expression. Previous studies have shown that loss of DHX9 protein increased circRNAs generation. These findings support the hypothesis that inorganic arsenic increases circRNAs expression by inhibiting DHX9 which is associated with promoter methylation. To test this hypothesis, we will examine the effects of arsenic on circRNAs and liner RNAs expression in peripheral blood lymphocytes among exposure group and control group and Pearson correlation coefficient will be used to explore associations between concentrations of three arsenic species in urine and gene expression. To further identify genes expression with aberrant DNA methylation, we will determine methylation levels by Methylation-sensitive high resolution melting. To investigate the efficacy of sodium arsenite, MMA, DNA and AZA in A549 cells，We will determined the RNA expression of NUP107, AKR1A1, DKC1and DHX9 after treatment for 5 days. To further identify genes expression with aberrant DNA methylation, we will assess the level of DHX9 gene DNA methylation with different concentrations of sodium arsenite. We will also exam the effects of arsenic, SAM and their combinations on the expression of RNA. To verify that the circRNAs production is dependent on DHX9 in the cells that have undergone arsenic exposure, the effect of siRNA-mediated DHX9 knockdown will be tested. This study will provide some potential for elucidating the underlying mechanisms of arsenic carcinogenesis.The study will provide evidence that circRNA can potentially be used as a biomarker of arsenic exposure.

CircRNAs是封闭环状结构的RNA与多种疾病相关。课题组研究显示亚砷酸钠和甲基化抑制剂（AZA）均诱导NUP107、AKR1A1和DKC1环状RNAs升高而对应线状RNA下降或不变，打破二者平衡。砷代谢物一甲基胂酸（MMA）、二甲基胂酸（DMA）并未引起类似效应。文献报道沉默DHX9使环状RNAs表达升高，我们发现亚砷酸钠和AZA可抑制DHX9表达。因此提出亚砷酸钠通过DNA甲基化抑制DHX9，DHX9下降导致环状RNAs表达增加，致使环状和线状RNAs比例失衡。因此使用亚砷酸钠、MMA、DMA和AZA作用细胞及收集砒霜厂高浓度砷暴露人群外周血淋巴细胞，检测上述基因环状和线状RNA表达及DHX9启动区DNA甲基化。接着砷联合甲基化供体作用细胞探索是否能逆转基因表达异常，最后沉默和过表达DHX9后暴露于砷，探索砷是否通过DHX9调控基因的环状RNA。为砷暴露防治与寻找生物标志物提供依据。

无机砷是一种常见的致癌性污染物，职业工人暴露于无机砷会导致肺癌、膀胱癌等疾病的高发。CircRNAs是封闭环状结构的一类RNA分子，在细胞的生长、分化、凋亡的过程中起重要作用，其表达变化与多种疾病相关。circRNA在血浆中能较为稳定，是具有生物标志物潜力的分子。DHX9是一个含有 DEAH 结构域的 RNA 解旋酶，通过与RNA上的Alu 元件结合，调控了大量circRNA的生成，在多种癌组织中表达升高。我们收集的砷暴露样本来自于砒霜厂其尿中的无机砷、一甲基胂酸和二甲基胂酸含量显著高于对照组与以往的研究是一致的。本课题组研究表明无机砷暴露会导致人群外周血淋巴细胞中DHX9mRNA减少，circRNAs(1017和2607)增加以及circRNA和mRNA比例（环线比）显著上调。在分子流行病研究中我们发现工人外周血淋巴细胞中的DHX9mRNA表达水平与环线比相关。工人尿中无机砷、一甲基胂酸和二甲基胂酸与工人外周血淋巴细胞中的DHX9mRNA成负相关，但是与circRNA(1017和2607)表达以及环线比成正相关。与砷代谢有的无机砷、一甲基胂酸和二甲基胂酸的百分含量也与上述也与上述三个RNA表达显著相关，最引人注明的是环线比与砷暴露的相关系数高于其他指标。我们采用A549和16HBE细胞研究发现砷暴露可以导致细胞中的DHX9mRNA下调以及 circRNAs(1017和2607)和环线比显著上调，与人群研究结果一致。细胞试验显示砷暴露导致了DHX9蛋白表达是上调，我们认为该结果是由于砷减弱了DHX9蛋白与MDM2的结合从而导致泛素化降解变化造成。课题组进一步通过siRNA下调DHX9的表达，结果显示2个细胞均表现为细胞活力明显下降，而凋亡显著上升。我们研究发现DHX9可以通过改变circRNA、mRNA的翻译、以及与蛋白的结合三个方面影响下游。DHX9的可以调控大量的有功能的circRNA生产变化。我们的结果还显示了P53途径的BIK、bax等基因表达显著上调，而NF-κB途径的CFLAR、Bcl-2达显著下调，DHX9可于与二者的mRNA和蛋白结合调控其翻译和激活。最后DHX9 还可以调控Bcl-X基因两个异构体Bcl-Xl和Bcl-Xs的比例进一步导致细胞凋亡。本研究提示了环线比有可能成为砷暴露效应标志物，以及DHX9在砷致癌中潜在的作用和机制提供了线索。