X射线诱导非小细胞肺癌A549细胞凋亡的适应性反应及相关miRNA筛选的研究

Adaptive response of NSCLC A549 cells induced by X-ray radiation and screening of related miRNAs

    摘要
  • 摘要:
    目的 研究X射线辐射诱导非小细胞肺癌（NSCLC）A549细胞凋亡的适应性反应，并筛选适应性反应相关的微RNA（miRNA）。
    方法 将NSCLC A549细胞分为6组，包括 50 mGy+20 Gy、200 mGy+20 Gy、20 Gy、50 mGy、200 mGy照射组及对照组（0 Gy），前2组细胞分别用50、200 mGy初始剂量进行照射，培养6 h后用20 Gy的效应剂量进行照射，20 Gy、50 mGy、200 mGy照射组同时进行照射。培养24 h后使用流式细胞仪检测细胞凋亡情况。利用小RNA测序技术筛选差异表达miRNA，并对其靶基因进行基因本体（GO）及京都基因与基因组百科全书（KEGG）通路的功能富集分析。采用实时荧光定量PCR（qRT-PCR）对部分差异表达miRNA进行验证。2组间数据的比较采用Welch t检验。
    结果 50 mGy+20 Gy照射组和200 mGy+20 Gy照射组的A549细胞早期凋亡率分别为（1.81±0.11）%和（2.17±0.19）%，低于20 Gy照射组的（4.54±0.23）%，且差异均有统计学意义（t=10.680、8.006，均P<0.01）。与20 Gy照射组相比，50 mGy+20 Gy照射组和200 mGy+20 Gy照射组共同差异表达趋势miRNA有1个上调（miR-3662）、15个下调（miR-185-3p、miR-1908-5p、miR-1307-5p、miR-182-3p、miR-92a-3p、miR-582-5p、miR-501-3p、miR-138-5P、miR-1260b、miR-484、miR-378d、miR-193b-3P、miR-127-3p、miR-1303及miR-654-5p）。GO富集分析结果显示，差异表达miRNA调控靶基因功能显著富集于细胞通讯调节、代谢过程的正向调节、代谢信号的调节、酶结合及催化活性等过程。KEGG富集分析结果显示，靶基因相关信号通路显著富集于溶酶体、丝裂原活化蛋白激酶、Ras和内吞作用等信号通路。qRT-PCR检测结果显示，miRNA表达情况与基因芯片结果趋势一致（10个miRNA表达水平得到验证）。
    结论 X射线50、200 mGy照射剂量均能诱导NSCLC A549细胞凋亡的适应性反应，并筛选到一组共同差异表达的miRNA，可能在X射线辐射诱导细胞凋亡的适应性反应中发挥了重要作用，有可能成为调节电离辐射生物效应的潜在靶点。

     

    Abstract:
    Objective To study the adaptive response of the X-ray radiation to apoptosis of non-small cell lung cancer (NSCLC) A549 cell, and to screen microRNAs (miRNAs) related to the adaptive effect of radiation.
    Methods NSCLC A549 cells were irradiated in 6 groups, namely, the50 mGy+20 Gy, 200 mGy+20 Gy, 20 Gy, 50 mGy, and 200 mGy irradiation groups and the 0 Gy as control group. The cells in the first two groups were irradiated with initial doses of 50 mGy and 200 mGy respectively, and were then irradiated with an effective dose of 20 Gy after culturs for 6 hours. The 20 Gy, 50 mGy, and 200 mGy groups were irradiated at the same time. The apoptosis of A549 cellswas detected by flow cytometry after 24 hours of culturing. Small RNA sequencing was performing to detect the expression of miRNAs in different irradiation groups, and quantitative real-time PCR (qRT-PCR) was used to verify some differentially expressed miRNAs. The target genes of differentially expressed miRNAs were predicted and then analyzed via gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses. The data between two groups were compared by performing Welch's t-test.
    Results The cell apoptosis rates of A549 cells in the 50 mGy+20 Gy and 200 mGy+20 Gy groups were (1.81±0.11)% and (2.17±0.19)% respectively, which were significantly lower than (4.54±0.23)% in the 20 Gy group (t=10.680, 8.006; all P<0.01). Meanwhile, the small RNA sequencing results show that compared with the 20 Gy irradiation group, 1 miRNA (miR-3662) was up-regulated and 15 miRNAs (miR-185-3p, miR-1908-5p, miR-1307-5p, miR-182-3p, miR-92a-3p, miR-582-5p, miR-501-3p, miR-138-5P, miR-1260b, miR-484, miR-378d, miR-193b-3P, miR-127-3p, miR-1303 and miR-654-5p) were down-regulated in the 50 mGy+20 Gy and 200 mGy+20 Gy irradiation groups. GO enrichment analysis reveal that the functions of target genes were significantly enriched in cell communication regulation, positive regulation of the metabolic process, regulation of the metabolic signal, enzyme binding and catalytic activity, whereas the KEGG analysis showed that the target genes related signal pathways were significantly enriched in lysosome, mitogen-activated protein kinase, Ras, endocytosis and other signal pathways. qRT-PCR was used to validate the expression of miRNAs, and the expression levels of 10 miRNAs were consistent with the sequencing results.
    Conclusions The X-ray of 50 mGy and 200 mGy radiation dose can induce an adaptive response in the cell apoptosis of A549 cells. A group of differently expressed miRNAs detected in 50 mGy+20 Gy and 200 mGy+20 Gy groups may play important roles in the adaptive response of low dose radiation and may become potential targets to regulate have effects of radiation.

     

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