六价铬职业接触人群血浆DNA损伤反应相关microRNA的鉴定及功能研究

Occupational exposure to hexavalent chromium (Cr (Ⅵ)) leads to various kinds of health damage by inducing DNA damage, but the underlying mechanims are not clear yet. Recent studies found that microRNA played an important role in regulating DNA damage response. Our previous study suggested that the expression levels of Plasma miR-340, miR-151, and miR-590 were up-regulated in workers occupationally exposed to Cr (Ⅵ), which indicated that microRNA might take part in DNA damage response induced by Cr (Ⅵ). Therefore, in the present study, single strand DNA breaks and double strand DNA breaks were studied in workers occupationally exposed to chromate and controls, and QuantiGene 80 Plex Assay was used to found differentially expressed genes related to DNA damage response; microRNA microarray is used to screen differential microRNAs in small sample size populations, and differentially expressed microRNAs related to DNA damage response will be confirmed after correlation analysis between differential genes and differential microRNAs, and the expression of differential miRNAs is validated in large sample size populations; The function of microRNAs is studied once their expression difference are confirmed, and the role of miRNAs in regulating DNA damage response induced by Cr (Ⅵ) is studied by reversing the expression of miRNAs, and the expression of target genes is validated by Dual Luciferase Reporter Gene Assay and rescuing assay. The aim of our study is to discover microRNAs and their target genes as biomarkers for workers occupationally exposed to Cr (Ⅵ), and to study the role of these microRNAs in response to DNA damage, providing a new road to mechanim study and prevention of the toxicity of Cr (Ⅵ).

职业接触六价铬可通过诱发DNA损伤致多种健康损害，但机制仍不清楚。研究显示，microRNA在调节DNA损伤反应中起重要作用。我们前期研究发现，铬盐职业接触工人血浆miR-340、miR-151和miR590的表达上调，提示miRNA参与六价铬诱导的DNA损伤反应。因此，本项目拟在铬盐职业接触人群中，用QuantiGene核酸定量技术检出DNA损伤反应相关差异基因；用芯片技术在小样本人群中筛出差异microRNAs，经关联分析后确定DNA损伤反应相关microRNAs，并用定量PCR方法在大样本人群中验证；在体外试验中，逆转表达后研究microRNAs在调控六价铬诱发DNA损伤反应中的作用，并用双荧光素酶报告基因法和回复试验验证靶基因。项目预期确定可作为铬盐职业接触人群生物标志物的microRNAs及靶基因，明确其在DNA损伤反应中的作用及机制，为六价铬毒作用的机制研究和预防提供新的思路。

本项目通过流式细胞术研究六价铬对人B淋巴母细胞系DNA损伤反应的影响，发现六价铬染毒可使细胞阻滞在G0/G1期，明显增加DNA损伤和细胞凋亡率。通过miRNA芯片比较六价铬染毒的人B淋巴母细胞染毒后24小时、去毒后3天和去毒后7天miRNA表达谱改变，发现三个时间点各有48、154和48个差异表达的miRNA。细胞验证试验中，去毒后3天共有miR-101-3p、miR-142-3p等9个差异表达的miRNA被验证。利用miRNA芯片筛测六价铬接触人群差异表达的miRNA，发现六价铬接触人群有45个差异表达的miRNA。人群验证结果中，共有9个miRNA差异表达的miRNA被验证。其中miR-424-3p人群验证结果与细胞验证结果表达趋势一致，miR-941的人群验证结果与人群筛测结果变化趋势一致。相关性分析显示miR-148a-3p等4个miRNA的表达与血铬浓度呈现负相关，miR-21-5p等3个miRNA的表达与血铬浓度呈现正相关，miR-19a-3p与暴露工龄之间成正相关。针对经筛选和验证的miRNAs，我们采用体外实验研究了miRNA在六价铬引起的DNA损伤反应中的作用，结果显示六价铬可通过抑制miR-148a-3p、miR-21-5p和促进miR-221-3p表达来抑制细胞凋亡，通过促进miR-424-3p表达来促进细胞凋亡。六价铬可通过抑制miR-148a-3p表达来抑制DNA损伤，通过促进miR-221-3p和miR-424-3p表达来促进DNA损伤。