Malinovskaya Elena M, Ershova Elizaveta S, Okorokova Natalya A, Veiko Vladimir P, Konkova Marina S, Kozhina Ekaterina A, Savinova Ekaterina A, Porokhovnik Lev N, Kutsev Serguey I, Veiko Nataly N, Kostyuk Svetlana V
Research Centre for Medical Genetics (RCMG), Moscow, Russia.
Biotechnology Research Center, Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
Front Oncol. 2019 May 30;9:445. doi: 10.3389/fonc.2019.00445. eCollection 2019.
The cell free ribosomal DNA (cf-rDNA) is accrued in the total pool of cell free DNA (cfDNA) in some non-cancer diseases and demonstrates DAMPs characteristics. The major research questions: (1) How does cell free rDNA content change in breast cancer; (2) What type of response in the MCF7 breast cancer cells is caused by cf-rDNA; and (3) What type of DNA sensors (TLR9 or AIM2) is stimulated in MCF7 in response to the action of cf-rDNA? CfDNA and gDNA were isolated from the blood plasma and the cells derived from 38 breast cancer patients and 20 healthy female controls. The rDNA content in DNA was determined using non-radioactive quantitative hybridization. In order to explore the rDNA influence on MCF7 breast cancer cells, the model constructs (GC-DNAs) were applied: pBR322-rDNA plasmid (rDNA inset 5836 bp long) and pBR322 vector. ROS generation, DNA damage, cell cycle, expression of TLR9, AIM2, NF-kB, STAT3, and RNA for 44 genes affecting the cancer cell viability were evaluated. The methods used: RT-qPCR, fluorescent microscopy, immunoassay, flow cytometry, and siRNA technology. The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, < 10). In MCF7, GC-DNAs induce a ROS burst, DNA damage response, and augmentation of NF-kB and STAT3 activity. The number of the apoptotic cells decreases, while the number of cells with an instable genome (G2/M- arrest, micronuclei) increase. Expression of anti-apoptotic genes () is elevated, while expression of pro-apoptotic genes () is lowered. The cells response for pBR322-rDNA is much more intense and develops much faster, than response for pBR322, and is realized through activation of TLR9- MyD88 - NF-kB- signaling. This difference in response speed is owing to the heightened oxidability of pBR322-rDNA and better ability to penetrate the cell. Induction of TLR9 expression in MCF7 is followed by blocking AIM2 expression. (1) Ribosomal DNA accumulates in cfDNA of breast cancer patients; (2) Cell free rDNA induce DNA damage response and stimulates cells survival, including cells with an instable genome; (3) Cell free rDNA triggers TLR9- MyD88- NF-kB- signaling, with significantly repressing the expression of AIM2.
在一些非癌症疾病中，游离核糖体DNA（cf-rDNA）会累积在游离DNA（cfDNA）的总库中，并表现出损伤相关分子模式（DAMP）的特征。主要研究问题如下：（1）乳腺癌中游离rDNA含量如何变化；（2）cf-rDNA会在MCF7乳腺癌细胞中引发何种类型的反应；（3）在MCF7中，cf-rDNA的作用会刺激何种类型的DNA传感器（TLR9或AIM2）？从38例乳腺癌患者和20名健康女性对照者的血浆及细胞中分离出cfDNA和基因组DNA（gDNA）。使用非放射性定量杂交法测定DNA中的rDNA含量。为了探究rDNA对MCF7乳腺癌细胞的影响，应用了模型构建体（GC-DNAs）：pBR322-rDNA质粒（插入片段为5836 bp长的rDNA）和pBR322载体。评估了活性氧（ROS）生成、DNA损伤、细胞周期、TLR9、AIM2、核因子κB（NF-κB）、信号转导和转录激活因子3（STAT3）的表达以及影响癌细胞活力的44个基因的RNA表达。所使用的方法包括：逆转录定量聚合酶链反应（RT-qPCR）、荧光显微镜检查、免疫测定、流式细胞术和小干扰RNA（siRNA）技术。病例组的R = cf-rDNA/g-rDNA比值高于对照组（中位数分别为3.4和0.8，<10）。在MCF7细胞中，GC-DNAs可诱导ROS爆发、DNA损伤反应，并增强NF-κB和STAT3的活性。凋亡细胞数量减少，而基因组不稳定的细胞（G2/M期阻滞、微核）数量增加。抗凋亡基因（）的表达升高，而促凋亡基因（）的表达降低。与对pBR322的反应相比，细胞对pBR322-rDNA的反应更为强烈且发展更快，并且是通过激活TLR9-MyD88-NF-κB信号通路实现的。这种反应速度的差异归因于pBR322-rDNA更高的氧化能力和更好的细胞穿透能力。在MCF7中诱导TLR9表达后会导致AIM2表达受阻。（1）核糖体DNA在乳腺癌患者的cfDNA中积累；（2）游离rDNA诱导DNA损伤反应并刺激细胞存活，包括基因组不稳定的细胞；（3）游离rDNA触发TLR9-MyD88-NF-κB信号通路，并显著抑制AIM2的表达。
