目的：探讨miR-339-5p通过调控Nudix结构水解酶5（Nudix hydrolase 5，NUDT5）的表达对肺癌A549细胞放射敏感 性的影响。方法：体外低浓度梯度递增结合大剂量间断冲击方法诱导产生耐X射线的肺癌A549细胞株（RA549），qPCR检测 miR-339-5p在人正常肺上皮细胞（BEAS-2B）、肺癌细胞系（A549、 L78、 H1299、 H460和RA549细胞）中的表达水平。根据对 RA549细胞的处理，实验分为NC组、 5 Gy组（5 Gy X 射线处理 RA549 细胞）、 5 Gy+miR-339-5p mimic组、 5 Gy+si-NUDT5组和 5 Gy+si-NUDT5+miR-339-5p inbibitor组，CCK-8法、Annexin V-FITC/PI双染流式术和WB分别检测各组细胞增殖、凋亡以及 NUDT5、 γ-H2AX 和 H2AX 蛋白表达的变化，双荧光素酶报告基因系统验证 miR-339-5p 和 NUDT5 的靶向关系。结果： miR-339-5p在各肺癌细胞系中表达显著低于BEAS-2B细胞（均P<0.05），其中RA549细胞表达水平最低。验证显示NUDT5是 miR-339-5p的靶基因。与 NC 组相比， 5 Gy 组 RA549 细胞增殖活力和 NUDT5 表达显著降低（均 P<0.01），凋亡率显著升 高（P<0.01）；与5 Gy组相比， 5 Gy+miR-339-5p mimic组RA549细胞的增殖活力显著降低（P<0.05）、凋亡率[（12.97±1.48） % vs （5.21±0.62） %， P<0.01]和γ-H2AX的表达水平（P<0.05）显 著 升 高 ， 5 Gy+si-NUDT5 组 RA549 细 胞 中 NUDT5 的表达水平 （P<0.01）和细胞增殖活力（P<0.01）均显著降低，凋亡率[（11.21±1.06） % vs（5.54±0.44） %， P<0.01]和γ-H2AX表达水平（P<0.01）均 显著升高， 而5 Gy+si-NUDT5+miR-339-5p inhibitor组细胞中上述指标均回复至5 Gy组水平。结论：过表达miR-339-5p通过靶 向下调NUDT5表达肺癌A549细胞的放射敏感性。

Objective: To explore the influence of miR-339-5p on the radio-sensitivity of lung cancer A549 cells by regulating the expression of Nudix hydrolase 5 (NUDT5). Methods: X-ray-resistant lung cancer A549 cells (RA549) were induced by treatment with low concentration gradient increment combined with large dose intermittent shock in vitro. The expression level of miR-339-5p in human normal lung epithelial cells (BEAS-2B) and lung cancer cell lines (A549, L78, H1299, H460 and RA549 cells) was detected by qPCR. According to the treatment, RA549 cells were divided into NC group, 5Gy group (treatment with 5Gy X-ray), 5Gy+miR-339-5p mimic group, 5Gy+si-NUDT5 group and 5Gy+si-NUDT5+miR-339-5p inhibitor group. CCK-8 assay,Annexin V-FITC/PI double staining flow cytometry and WB were used to detect the proliferation, apoptosis and the protein expressions of NUDT5, γ-H2AX and H2AX in each group. The targeting relationship between mir-339-5p and NUDT5 was detected by Dual-luciferase reporter gene system. Results: The expression of miR-339-5p in lung cancer cell lines was significantly lower than that in BEAS-2B cells, with the lowest ex- pression level in RA549 cells (all P<0.05). NUDT5 was the target gene of miR-339-5p. Compared with the NC group, the proliferation activity and NUDT5 expression of RA549 cells in the 5 Gy group were significantly reduced (all P<0.01), and the apoptosis rate was significantly increased (P<0.01). Compared with the 5 Gy group, the proliferation activity of RA549 cells in the 5 Gy+miR-339-5p mimic group was significantly reduced (P<0.05), the apoptosis rate ([12.97±1.48]% vs [5.21±0.62]%, P<0.01) and the expression level of γ-H2AX (P<0.05) were significantly increased; the expression of NUDT5 (t=7.58, P<0.01) and cell proliferation activity (t=6.58, P< 0.01) of RA549 cells in the 5 Gy+si-NUDT5 group were significantly reduced, while the apoptosis rate ([11.21±1.06]% vs [5.54± 0.44%, P<0.01) and the expression of γ-H2AX (P<0.01) were significantly increased; and the above indicators in 5 Gy+si-NUDT5+ miR-339-5p inhibitor group showed insignificant difference from the 5 Gy group. Conclusion: Overexpression of miR-339-5p enhances the radio-sensitivity of X-ray-resistant lung cancerA549 cells by targetedly down-regulating NUDT5 expression.

国家重点基础研究发展（973计划）计划资助项目（No. 2014CB542102）；国家自然科学基金资助项目（No. 31570869；31170844）