核糖体DNA拷贝数变异在六价铬诱发DNA损伤反应中的作用及机制研究

Occupational exposure to hexavalent chromium (Cr (Ⅵ)) can lead to health injury including lung cancer through DNA damage, but its mechanisms are uncertain till now. Previous studies showed that ribosomal DNA (rDNA) might play an important role in DNA damage response (DDR). Our recent studies found rDNA copy number variation (CNV) after Cr (Ⅵ) treatment in cell line, SD rats, and food flies. Which indicated that rDNA might be involved in DDR induced by Cr (Ⅵ). In the present study, the specificity of rDNA CNV induced by Cr (Ⅵ) will be confirmed in vitro. The involvement of rDNA CNV in regulating DDR will be investigated through dynamic changes analysis between rDNA CNV and DDR, and analyzing the effects of rDNA CNV on DDR induced by Cr (Ⅵ) in the same cell line and between different cell lines. The nucleolar proteins taking part in the regulation of DDR induced by Cr (Ⅵ) will be selected by RNA sequencing for screening and by liquid suspension chip for validating. The roles of candidate genes in Cr (Ⅵ)-induced DDR will be studied using gene editing technique. Finally, in order to analyze the possibility of rDNA copy number as a biomarker of effect in workers occupationally exposed to hexavalent chromium, the relationship between rDNA CNV and chromium exposure levels, DNA damage, and the expression levels of related genes will be investigated in the population studies, including cross-sectional study and dynamic follow-up study. The project is expected to provide a new way to study the toxicity of chromium, and to provide sensitive biomarkers for workers occupationally exposed to chromate.

职业接触六价铬可通过DNA损伤诱发肺癌等健康损害，但机制仍未明确。研究显示核糖体DNA（rDNA）在DNA损伤反应中起重要作用。我们前期研究发现六价铬在体内外实验中均可致rDNA拷贝数变异，提示其可能参与六价铬诱导的DNA损伤反应。因此，本项目拟首先确定六价铬诱发的rDNA拷贝数变异具有特异性；然后经动态变化关系分析、rDNA拷贝数变异对六价铬诱发DNA损伤反应的影响研究，明确rDNA拷贝数变异参与调控DNA损伤反应；再采用RNA测序和液相悬浮芯片技术筛选并验证参与DNA损伤反应调控的核仁蛋白，并用基因编辑的方式研究其可能的调控机制；最后，通过铬盐接触人群和对照的比较及新上岗工人的动态随访研究rDNA拷贝数变异与六价铬负荷、接触时间、DNA损伤及相关基因表达之间的关系，明确其作为效应标志物的可能性。项目可为六价铬毒作用机制研究提供新思路，为铬盐职业接触人群提供候选生物标志物。

本项目通过实时荧光定量PCR和流式细胞术确定六价铬暴露水平对rDNA拷贝数变异和DNA损伤反应的影响，并根据前期RNA测序技术以及结合rDNA 拷贝数动态变化筛选出的与之相关的核仁蛋白基因，采用蛋白质免疫印迹实验对差异基因进行蛋白表达验证，研究相应核仁蛋白基因敲除后六价铬诱发的rDNA 拷贝数及其与 DDR 之间的关系，确认相应核仁蛋白基因参与调控的 DDR 过程。体外研究中，我们分别在不同细胞系（人B淋巴母细胞HMy2.CIR、人正常肺上皮细胞BEAS-2B、人胚肺细胞MRC-5、人宫颈癌细胞Hela）中研究 rDNA 拷贝数 变异对六价铬诱发 DDR 的影响。并通过前期转录组测序结果选择了40个DNA损伤反应相关核仁蛋白，其中ZNF385A、UBA52、HRAS这3个蛋白转录水平的变化经体外验证和rDNA拷贝数变化趋势一致。进一步研究发现，HRAS、ZNF385A转录水平抑制可致六价铬诱发的rDNA拷贝数升高、DNA损伤及细胞凋亡效应更明显。UBA52转录水平的抑制可使细胞阻滞在G2/M期。人群研究验证中发现，六价铬暴露可致职业人群血液基因组rDNA拷贝数升高，且与血铬浓度呈正相关。六价铬职业暴露人群HRAS、ZNF385A转录水平、蛋白水平、rDNA拷贝数、总DNA损伤水平均升高，HRAS转录水平与尿铬浓度呈负相关，与45S rDNA拷贝数呈正相关，与ATM激活呈负相关。人群ZNF385A转录水平与45SrDNA拷贝数，DNA双链断裂，H2AX磷酸化、总DNA损伤呈显著性正相关，ZNF385A转录水平与蛋白水平呈显著性正相关。人群UBA52转录水平与DNA双链断裂和总DNA损伤呈显著性正相关，UBA52蛋白水平与ATM激活呈显著性正相关。UBA52转录水平与蛋白水平呈显著性正相关。

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