Abstract
Introduction: 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?Materials and Methods: 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.Results: The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, p < 10−8). 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 (BCL2, BCL2A1, BCL2L1, BIRC3, MDM2) is elevated, while expression of pro-apoptotic genes (BAX, BID, BAD, PMAIP1, BBC3) 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.Conclusion: (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.
Highlights
The cell free ribosomal DNA is accrued in the total pool of cell free DNA in some non-cancer diseases and demonstrates DAMPs characteristics
transcribed region of human ribosomal repeat (TR-rDNA) Is Accumulated in Circulating cell-free DNA (cfDNA) of Breast Cancer Patients
Circulating cfDNA and gDNA were isolated from blood plasma and leukocytes of 38 female breast cancer patients, who received radiation therapy or chemotherapy, and 20 healthy female controls
Summary
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 PRRs recognize foreign molecules of viruses and bacteria These sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines [1]. The products of damaged cells, which are recognized by PRRs, are termed alarmins, or damage-associated molecular pattern (DAMPs) signals. Nucleic acids, which are released by damaged and dying cells, are recognized by some PRRs and belong to the DAMPs family [4, 5]. These DAMPs enter into the composition of cell-free DNA (cfDNA) and underlie the biological activity of cfDNA
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