Objective To explore the differential gene expression profiling of 16-HBE cells under Cr(Ⅵ) exposure. The expressions of differentially expressed genes related to DNA damage repairing were analyzed and key genes in the production of this toxic effect was screen, providing novel perspective and theoretical basis for further study on toxicological mechanisms of hexavalent chromium.
Methods The profiling of differential gene expression induced by acute Cr(Ⅵ) exposure in 16-HBE cellswas established by gene chip (Affymetrix); bioinformatics methods were used to analyze the corresponding biological functions of differentially expressed genes, and the expression levels of differentially expressed genes were further verified by Western blot.
Results The results of gene chip revealed more than 2-folds expression variations in nearly 200 genes. Bioinformatics analysis indicated that differentially expressed genes were mainly aggregated at pathways related to DNA damage repairing, transcription and translation; the elevated expressions of genes related to DNA damage repairing including FEN1, PCNA and APX1, among which the expression of FEN1 and PCNA genes increased by about 100%, and the difference was statistically significant ( P<0.01). The results were consistent with gene chip screening, and consolidated by Western blot which showed the elevated expression of FEN1, PCNA and APX1 at protein level, among them FEN1 was significantly increased ( P<0.05).
Conclusion The pathway related to DNA damage repair was activated and the expression of DNA damage repair gene FEN1 was up-regulated in 16-HBE cells underwent acute Cr(VI) exposure, suggesting that the pathway play an important role in Cr(VI)-associated toxicity.
摘要： 目的 建立六价铬 C (r Ⅵ) 暴露人支气管上皮细胞 (16HBE) 的基因差异表达谱, 并分析其致 DNA 损伤修复相 关差异基因表达改变, 筛选该毒性作用产生过程中的关键基因, 为研究六价铬毒性机制提供新的思路和理论依据。 方法 采用 Affymetrix 3 ′ IVT 基因芯片检测 Cr(Ⅵ) 急性暴露 16HBE 细胞诱导的差异基因表达谱, 运用生物信息学方法 分析差异基因的相关生物学功能; 进一步用 Western blot 蛋白电泳技术验证差异基因的蛋白表达水平。 结果 Affyme⁃ trix 3 ′ IVT 基因芯片结果显示约 200 个基因表达变化大于 2 倍以上; 生物信息学分析提示差异基因主要富集在 DNA 损 伤与修复、转录与翻译等通路; DNA 损伤修复通路中, FEN1、PCNA 和 APX1 的 mRNA 表达水平均呈上升趋势, 其中 FEN1 和 PCNA 基 因 的 表 达 量 均 增 加 了 约 100%, 差 异 有 统 计 学 意 义 ( P<0.01); Western blot 结 果 显 示 FEN1、PCNA 和 APX1 表达水平均呈上升趋势, 与基因表达变化一致, 其中 FEN1 的表达变化差异有统计学意义 ( P<0.05) 。 结论 C (r Ⅵ) 急性暴露人支气管上皮细胞可激活 DNA 损伤修复信号通路, 使 DNA 损伤修复基因 FEN1 表达上调, DNA 损伤修复通路 可能在 C (r VI) 诱导 16HBE 细胞毒性中发挥重要作用。