长链非编码RNA在六价铬诱导DNA损伤修复中的功能和调控机制研究

Hexavalent chromium [Cr (Ⅵ)] is a well recognized human carcinogen. It can cause serious environmental pollution, the hazards involved in the occupational and general population. Cr (Ⅵ) can cause DNA double-strand breaks (DSB) and damage. However, the molecular mechanism of Cr (Ⅵ) caused human cancer are still not well understood. Many investigation and studies showed that the epigenetic modification plays an important role in homologous recombination (HR) of DSB. Our previous studies found that Cr (Ⅵ) can induce a differentially expression of long non-coding RNA (lncRNA) ENST00000526635 and NR_040058 significantly in human bronchial epithelial cells (16hBE) treated with Cr (Ⅵ). The bioinformatics analysis revealed that the two lncRNA above is closely related to the key protein RAD51 in DSB HR. This result suggested that the ENST00000526635 and NR_040058 may play an important regulatory function in HR of DSB induced by Cr (Ⅵ). Up to now, there are have not any studies on the mechanisms and functions of lncRNA in repair of DSB caused by Cr (Ⅵ) exposure. Based on our preliminary work, we will analyze the regulatory roles of ENST00000526635 and NR_040058 on the signaling pathways and potential target genes of HR during DSB caused by Cr (Ⅵ) exposure, further study the function and regulation mechanism of them in the HR of DSB caused by Cr (Ⅵ). Our project is helpful not only for clarification of the function and regulation molecule mechanisms of lncRNA in the repair of DSB caused by Cr (Ⅵ), but also contributes to find biomarkers of DNA damage repair in Cr (Ⅵ) exposure and the prevention carcinogen of Cr (Ⅵ).

六价铬[Cr（Ⅵ）]可导致DNA损伤和双链断裂（DSB），其属于人Ⅰ类致癌物。Cr（Ⅵ）造成的环境污染可严重危害职业人群和普通居民的健康。研究显示表观遗传修饰在DSB同源重组修复（HR）中具有重要功能。我们预实验显示Cr （Ⅵ）可诱导人支气管上皮细胞长链非编码RNA（lncRNA）ENST00000526635和NR_040058显著差异表达，其与DSB HR中的关键蛋白RAD51密切相关，由此我们假设上述2个lncRNA可通过调控HR在Cr（Ⅵ）诱导的DSB修复中发挥作用。本项目拟在前期工作基础上，研究以上lncRNA的功能及其对Cr（Ⅵ）暴露诱导DSB HR调控的分子机制，观察其在铬暴露工人外周血PBMC中的表达与Cr（Ⅵ）的关系。本课题不仅对阐明表观遗传修饰在Cr(Ⅵ)诱导DSB修复中的功能和作用机制，而且对寻找Cr（Ⅵ）暴露DNA损伤修复生物标志物及其遗传毒性的防控均有重要作用。

六价铬[Cr（Ⅵ）]可导致DNA损伤和双链断裂（DSB）。研究显示lncRNA 在维持基因组稳定中发挥重要作用，但关于lncRNA 对Cr（Ⅵ）暴露诱导DSB 修复中变化及作用机制尚有待阐明。本研究通过基因芯片获取Cr (Ⅵ)染毒BEAS-2B时lncRNA差异表达谱，采用RT-qPCR对选定lncRNA进行验证，结合生物信息学分析筛选lncRNA；用慢病毒质粒构建lncRNA过表达载体、小干扰RNA构建lncRNA敲减模型，CCK8法测定细胞增殖活性，间接免疫荧光法测定DNA损伤及修复指标γH2AX foci和RAD51 foci阳性细胞%，RNA pulldown分析与选定差异表达lncRNA结合的蛋白和RNA。结果显示Cr (Ⅵ)染毒BEAS-2B细胞后lncRNA具有明显差异表达，这些差异lncRNA主要富集在肿瘤、翻译、信号转导及代谢通路；共表达网络分析显示lnc-DHFR-4:1、lnc-ERCC6L-2:1、NR_110023、ENST00000599817、ENST00000607815等lncRNA可能在Cr (Ⅵ)诱导DNA损伤修复中发挥重要作用。根据lncRNA功能预测选取lnc-DHFR-4:1作为研究对象。荧光原位杂交实验显示lnc-DHFR-4:1主要分布胞质；lnc-DHFR-4:1过表达使细胞增殖活性显著增加，而敲减可导致细胞增殖活性显著下降，提示lnc-DHFR-4:1对Cr (Ⅵ)诱导的细胞毒具有保护作用。过表达lnc-DHFR-4:1后γH2AX foci阳性细胞%显著降低，RAD51 foci阳性细胞%显著增加；提示lnc-DHFR-4:1可影响细胞增殖，通过γH2AX foci和RAD51 foci途径影响细胞DNA损伤的修复；RNA pulldown分析显示lnc-DHFR-4:1可多种蛋白和RNA（LncRNA、circRNA和mRNA）结合，相关蛋白和RNA涉及多个信号通路和细胞生物学功能。现场研究观察铬盐暴露工人外周血单个核细胞DNA断裂损伤、尿8-OHdG、全DNA甲基化、PDCD5表达，探讨Cr (Ⅵ)暴露诱导细胞凋亡与DNA损伤、DNA甲基化的相关性。从不同角度和层面对铬酸盐所致的遗传损伤与表观遗传学变化，为铬盐暴露所致遗传毒性机制研究奠定基础。