medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.25557/2073-7998.2021.08.48-59medgen-1962Research ArticleОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯORIGINAL RESEARCHИсследование с применением системы редактирования генома CRISPR/Cas9 биологического действия ГЦ-богатых последовательностей внеклеточной ДНК на процесс регуляции транскрипционной активности ДНК-сенсоров TLR9 и AIM2 в клетках линии MCF7Genome editing technology CRISPR/Cas9 in the study of the biological effect of GC-rich cell-free DNA sequences (cfDNA) on the regulation of the transcriptional activity of TLR9 receptor-DNA sensor genes and AIM2 on the MCF7 line cellsМалиновскаяЕ. М.MalinovskayaE. M.m.elena.0402@gmail.comКожинаЕ. А.KozhinaE. A.noemail@neicon.ruЕршоваЕ. С.ErshovaE. S.noemail@neicon.ruКоньковаМ. С.KonkovaM. S.noemail@neicon.ruВейкоВ. П.VeikoV. P.noemail@neicon.ruБобровскийП. А.BobrovskyP. A.noemail@neicon.ruЛазаревВ. Н.LazarevV. N.noemail@neicon.ruШмаринаГ. В.ShmarinaG. V.noemail@neicon.ruКаменеваЛ. В.KamenevaL. V.noemail@neicon.ruВейкоН. Н.VeikoN. N.noemail@neicon.ruКостюкС. В.KostyukS. V.noemail@neicon.ruФГБНУ «Медико-генетический научный центр имени академика Н.П. Бочкова»РоссияResearch Centre for Medical GeneticsRussian FederationФГБНУ «Медико-генетический научный центр имени академика Н.П. Бочкова»; ФИЦ «Фундаментальные основы биотехнологии Российской академии наук»РоссияResearch Centre for Medical Genetics; Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of SciencesRussian FederationФГБУ ФНКЦ Физико-химической медицины ФМБА РоссииРоссияFederal Research and Clinical Center of physical-chemical medicine of Federal Medical Biological AgencyRussian Federation2021231120212084859Copyright © Малиновская Е.М., Кожина Е.А., Ершова Е.С., Конькова М.С., Вейко В.П., Бобровский П.А., Лазарев В.Н., Шмарина Г.В., Каменева Л.В., Вейко Н.Н., Костюк С.В., 20212021Малиновская Е.М., Кожина Е.А., Ершова Е.С., Конькова М.С., Вейко В.П., Бобровский П.А., Лазарев В.Н., Шмарина Г.В., Каменева Л.В., Вейко Н.Н., Костюк С.В.Malinovskaya E.M., Kozhina E.A., Ershova E.S., Konkova M.S., Veiko V.P., Bobrovsky P.A., Lazarev V.N., Shmarina G.V., Kameneva L.V., Veiko N.N., Kostyuk S.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.medgen-journal.ru/jour/article/view/1962

Ряд заболеваний, в том числе онкологических, сопровождается повышенным уровнем накопления ГЦ-обогащенных последовательностей в общем пуле свободной внеклеточной ДНК (вкДНК). Во многом этот факт объясняется повышенной устойчивостью этих фрагментов ДНК к действию нуклеаз крови. Они могут активировать ДНК-сенсоры нуклеиновых кислот - TLR9 и AIM2, которые играют существенную роль в функционировании раковых клеток. Цель исследования: изучение биологического ответа клеток линии MCF7 на действие ГЦ-богатых последовательностей вкДНК и выявление с помощью нокаута генов AIM2 и TLR9 функциональной роли ДНК-связывающих рецепторов в развитии клеточного адаптивного ответа. Экспериментально установлено, что клетки культуры MCF7 с «выключенными» рецепторами TLR9 и AIM2 отвечают на стимуляцию фрагментами ГЦ-богатых последовательностей вкДНК снижением транскрипционной активности генов сигнальных каскадов TLR9/MYD88/NF-kB-сигнального пути и связанных с ним STAT 3/6-сигнальных путей, повышающих выживаемость раковых клеток. Это показывает необходимость дальнейших исследований роли других генов в раковых клетках для детализации механизмов отмеченных в данной работе эффектов по выживаемости раковых клеток.

A number of diseases, including cancer, are associated with accumulation of GC-enriched sequences in the general cell-free DNA (cfDNA) pool. This fact is primarily explained by these DNA fragments increased resistance to the blood nucleases which can activate DNA sensors - TLR9 and AIM2 that have a different effect on the cancer cells functioning. The aim of the present study was to study the MCF 7 cells biological response to GC-rich cfDNA sequences and using AIM2 and TLR9 knockout to identify the functional role of DNA-binding receptors in the development of cellular adaptive response. It was experimentally established that MCF7 culture cells with TLR9 and AIM2 receptors “turned off” respond to stimulation by GC-DNA fragments by reducing the transcriptional activity of the genes of the TLR9/MYD88/NF-KBsignaling pathway and associated STAT 3/6 signaling pathways that increase the survival of cancer cells. This shows the need for further studies of the role of other genes, including on the example of cell lines with TLR9 and AIM2 knockout, in terms of detailing the mechanisms of the effects noted in this work on cancer cell survival.

ГЦ-вкДНКклеточная линия MCF7генные регуляторные сетинокаут генов TLR9 и AIM2CRISPR/Cas9 технологияGC-cfDNAcell line MCF7gene regulatory networksgene knockoutCRISPR/Cas9 technologyReferencesStewart C.M., Kothari P.D., Mouliere F. et al. The value of cell-free DNA for molecular pathology. J Pathol. 2018 Apr;244(5):616-627.Stewart C.M., Kothari P.D., Mouliere F. et al. The value of cell-free DNA for molecular pathology. J Pathol. 2018 Apr;244(5):616-627.Niu Z., Tang W., Liu T. et al. Cell-free DNA derived from cancer cells facilitates tumor malignancy through Toll-like receptor 9 signaling-triggered interleukin-8 secretion in colorectal cancer. Acta Biochim Biophys Sin (Shanghai). 2018 Oct 1;50(10):1007-1017.Niu Z., Tang W., Liu T. et al. Cell-free DNA derived from cancer cells facilitates tumor malignancy through Toll-like receptor 9 signaling-triggered interleukin-8 secretion in colorectal cancer. Acta Biochim Biophys Sin (Shanghai). 2018 Oct 1;50(10):1007-1017.Jahr S., Hentze H., Englisch S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001 Feb 15;61(4):1659-65.Jahr S., Hentze H., Englisch S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001 Feb 15;61(4):1659-65.Spindler K.G., Boysen A.K., Pallisgård N. et al. Cell-free DNA in metastatic colorectal cancer: a systematic review and meta-analysis. Oncologist. 2017 Sep;22(9):1049-1055.Spindler K.G., Boysen A.K., Pallisgård N. et al. Cell-free DNA in metastatic colorectal cancer: a systematic review and meta-analysis. Oncologist. 2017 Sep;22(9):1049-1055.Suzuki N., Kamataki A., Yamaki J., Homma Y. Characterization of circulating DNA in healthy human plasma. Clin Chim Acta. 2008 Jan;387(1-2):55-8.Suzuki N., Kamataki A., Yamaki J., Homma Y. Characterization of circulating DNA in healthy human plasma. Clin Chim Acta. 2008 Jan;387(1-2):55-8.Lander E.S., Linton L.M., Birren B. et al. Initial sequencing and analysis of the human genome. Nature. 2001 Feb 15;409(6822):860-921.Lander E.S., Linton L.M., Birren B. et al. Initial sequencing and analysis of the human genome. Nature. 2001 Feb 15;409(6822):860-921.Kozhina E.A., Ershova E.S., Okorokova N.A. et al. Extracellular DNA containing (dG)n motifs penetrates into MCF7 breast cancer cells, induces the adaptive response, and can be expressed. Oxid Med Cell Longev. 2019 Nov 3;2019:7853492.Kozhina E.A., Ershova E.S., Okorokova N.A. et al. Extracellular DNA containing (dG)n motifs penetrates into MCF7 breast cancer cells, induces the adaptive response, and can be expressed. Oxid Med Cell Longev. 2019 Nov 3;2019:7853492.Alvarado-Vásquez N. Circulating cell-free mitochondrial DNA as the probable inducer of early endothelial dysfunction in the prediabetic patient. Exp Gerontol. 2015 Sep;69:70-8.Alvarado-Vásquez N. Circulating cell-free mitochondrial DNA as the probable inducer of early endothelial dysfunction in the prediabetic patient. Exp Gerontol. 2015 Sep;69:70-8.Tuboly E., Mcllroy D., Briggs G. et al. Clinical implications and pathological associations of circulating mitochondrial DNA. Front Biosci (Landmark Ed). 2017 Jan 1;22:1011-1022.Tuboly E., Mcllroy D., Briggs G. et al. Clinical implications and pathological associations of circulating mitochondrial DNA. Front Biosci (Landmark Ed). 2017 Jan 1;22:1011-1022.Вейко Н.Н., Булычева Н.В., Рогинко О.А., Вейко Р.В., Ершова Е.С., Коздоба О.А., Кузьмин В.А., Виноградов А.М., Юдин А.А., Сперанский А.И. Фрагменты транскрибируемой области рибосомного повтора в составе внеклеточной ДНК - маркер гибели клеток организма. Биомедицинская химия 2008; 54(1): 78-93.Вейко Н.Н., Булычева Н.В., Рогинко О.А., Вейко Р.В., Ершова Е.С., Коздоба О.А., Кузьмин В.А., Виноградов А.М., Юдин А.А., Сперанский А.И. Фрагменты транскрибируемой области рибосомного повтора в составе внеклеточной ДНК - маркер гибели клеток организма. Биомедицинская химия 2008; 54(1): 78-93.Veiko N.N., Shubaeva N.O., Ivanova S.M. et al. Blood serum DNA in patients with rheumatoid arthritis is considerably enriched with fragments of ribosomal repeats containing immunostimulatory CpG-motifs. Bull Exp Biol Med. 2006 Sep;142(3):313-6.Veiko N.N., Shubaeva N.O., Ivanova S.M. et al. Blood serum DNA in patients with rheumatoid arthritis is considerably enriched with fragments of ribosomal repeats containing immunostimulatory CpG-motifs. Bull Exp Biol Med. 2006 Sep;142(3):313-6.Вейко, Н. Н., Конорова, И. Л., Неверова, М. Е., Фиделина, О. В., Мкртумова, Н. А., Ершова, Е. С., Постнов, А. Ю., Конькова, М. С. Влияние CpG - богатых фрагментов ДНК на формирование гипертензии у спонтанно-гипертензивных крыс (SHR). Биомедицинская химия 2010; 56(6): 686-699.Вейко, Н. Н., Конорова, И. Л., Неверова, М. Е., Фиделина, О. В., Мкртумова, Н. А., Ершова, Е. С., Постнов, А. Ю., Конькова, М. С. Влияние CpG - богатых фрагментов ДНК на формирование гипертензии у спонтанно-гипертензивных крыс (SHR). Биомедицинская химия 2010; 56(6): 686-699.Korzeneva I.B., Kostuyk S.V., Ershova E.S. et al. Human circulating ribosomal DNA content significantly increases while circulating satellite III (1q12) content decreases under chronic occupational exposure to low-dose gamma- neutron and tritium beta-radiation. Mutat Res. 2016 Sep - Oct;791-792:49-60.Korzeneva I.B., Kostuyk S.V., Ershova E.S. et al. Human circulating ribosomal DNA content significantly increases while circulating satellite III (1q12) content decreases under chronic occupational exposure to low-dose gamma- neutron and tritium beta-radiation. Mutat Res. 2016 Sep - Oct;791-792:49-60.Честков И.В., Вейко Н.Н., Ершова Е.С., Сергеева В.А., Вейко Р.В., Ижевская В.Л., Костюк С.В. Метод анализа числа копий GC-богатых повторяющихся последовательностей генома в составе поврежденной ДНК. Определение увеличенного содержания рибосомных генов в циркулирующей внеклеточной ДНК лиц с длительным стажем курения табака. Медицинская генетика. 2016;15(1):43-50.Честков И.В., Вейко Н.Н., Ершова Е.С., Сергеева В.А., Вейко Р.В., Ижевская В.Л., Костюк С.В. Метод анализа числа копий GC-богатых повторяющихся последовательностей генома в составе поврежденной ДНК. Определение увеличенного содержания рибосомных генов в циркулирующей внеклеточной ДНК лиц с длительным стажем курения табака. Медицинская генетика. 2016;15(1):43-50.Malinovskaya E.M., Ershova E.S., Okorokova N.A. et al. Ribosomal DNA as DAMP signal for MCF7 cancer cells. Front Oncol. 2019 May 30;9:445.Malinovskaya E.M., Ershova E.S., Okorokova N.A. et al. Ribosomal DNA as DAMP signal for MCF7 cancer cells. Front Oncol. 2019 May 30;9:445.Kostyuk S.V., Konkova M.S., Ershova E.S. et al. An exposure to the oxidized DNA enhances both instability of genome and survival in cancer cells. PLoS One. 2013 Oct 17;8(10):e77469.Kostyuk S.V., Konkova M.S., Ershova E.S. et al. An exposure to the oxidized DNA enhances both instability of genome and survival in cancer cells. PLoS One. 2013 Oct 17;8(10):e77469.Kostyuk S., Smirnova T., Kameneva L. et al. GC-rich extracellular DNA induces oxidative stress, double-strand DNA breaks, and DNA damage response in human adipose-derived mesenchymal stem cells. Oxid Med Cell Longev. 2015;2015:782123.Kostyuk S., Smirnova T., Kameneva L. et al. GC-rich extracellular DNA induces oxidative stress, double-strand DNA breaks, and DNA damage response in human adipose-derived mesenchymal stem cells. Oxid Med Cell Longev. 2015;2015:782123.He L., Liu Y., Lai W. et al. DNA sensors, crucial receptors to resist pathogens, are deregulated in colorectal cancer and associated with initiation and progression of the disease. J Cancer 2020; 11(4):893-905.He L., Liu Y., Lai W. et al. DNA sensors, crucial receptors to resist pathogens, are deregulated in colorectal cancer and associated with initiation and progression of the disease. J Cancer 2020; 11(4):893-905.Li T.T., Ogino S., Qian Z.R. Toll-like receptor signaling in colorectal cancer: carcinogenesis to cancer therapy. World J Gastroenterol. 2014;20:17699-708.Li T.T., Ogino S., Qian Z.R. Toll-like receptor signaling in colorectal cancer: carcinogenesis to cancer therapy. World J Gastroenterol. 2014;20:17699-708.Sipos.F, Furi I., Constantinovits M., Tulassay Z., Muzes G. Contribution of TLR signaling to the pathogenesis of colitis-associated cancer in inflammatory bowel disease. World J Gastroenterol. 2014;20:12713-21.Sipos.F, Furi I., Constantinovits M., Tulassay Z., Muzes G. Contribution of TLR signaling to the pathogenesis of colitis-associated cancer in inflammatory bowel disease. World J Gastroenterol. 2014;20:12713-21.Man S.M., Zhu Q., Zhu L. et al. Critical role for the DNA sensor AIM2 in stem cell proliferation and cancer. Cell. 2015;162:45-58.Man S.M., Zhu Q., Zhu L. et al. Critical role for the DNA sensor AIM2 in stem cell proliferation and cancer. Cell. 2015;162:45-58.Chen J., Wang Z., Yu S. AIM2 regulates viability and apoptosis in human colorectal cancer cells via the PI3K/Akt pathway. Onco Targets Ther. 2017;10:811-7.Chen J., Wang Z., Yu S. AIM2 regulates viability and apoptosis in human colorectal cancer cells via the PI3K/Akt pathway. Onco Targets Ther. 2017;10:811-7.Hemmi H., Takeuchi O., Kawai T. et al. A Toll-like receptor recognizes bacterial DNA. Nature. 2000 Dec 7;408(6813):740-5.Hemmi H., Takeuchi O., Kawai T. et al. A Toll-like receptor recognizes bacterial DNA. Nature. 2000 Dec 7;408(6813):740-5.Yasuda K., Richez C., Uccellini M.B. et al. Requirement for DNA CpG content in TLR9-dependent dendritic cell activation induced by DNA-containing immune complexes. J Immunol. 2009 Sep 1;183(5):3109-17.Yasuda K., Richez C., Uccellini M.B. et al. Requirement for DNA CpG content in TLR9-dependent dendritic cell activation induced by DNA-containing immune complexes. J Immunol. 2009 Sep 1;183(5):3109-17.Väisänen M.R., Jukkola-Vuorinen A., Vuopala K.S., Selander K.S., Vaarala M.H. Expression of Toll-like receptor-9 is associated with poor progression-free survival in prostate cancer. Oncol Lett. 2013 May;5(5):1659-1663.Väisänen M.R., Jukkola-Vuorinen A., Vuopala K.S., Selander K.S., Vaarala M.H. Expression of Toll-like receptor-9 is associated with poor progression-free survival in prostate cancer. Oncol Lett. 2013 May;5(5):1659-1663.Zhang Y., Wang Q., Ma A., Li Y., Li R., Wang Y. Functional expression of TLR9 in esophageal cancer. Oncol Rep. 2014 May;31(5):2298-304.Zhang Y., Wang Q., Ma A., Li Y., Li R., Wang Y. Functional expression of TLR9 in esophageal cancer. Oncol Rep. 2014 May;31(5):2298-304.Herrmann A., Cherryholmes G., Schroeder A. et al. TLR9 is critical for glioma stem cell maintenance and targeting. Cancer Res. 2014 Sep 15;74(18):5218-28.Herrmann A., Cherryholmes G., Schroeder A. et al. TLR9 is critical for glioma stem cell maintenance and targeting. Cancer Res. 2014 Sep 15;74(18):5218-28.Barrat F.J., Meeker T., Gregorio J. et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med. 2005 Oct 17;202(8):1131-9.Barrat F.J., Meeker T., Gregorio J. et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med. 2005 Oct 17;202(8):1131-9.Wang W., Kong P., Ma G. et al. Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget. 2017 Jun 27;8(26):43180-43191.Wang W., Kong P., Ma G. et al. Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget. 2017 Jun 27;8(26):43180-43191.Sharma B.R., Karki R., Kanneganti T.D. Role of AIM2 inflammasome in inflammatory diseases, cancer and infection. Eur J Immunol. 2019 Nov;49(11):1998-2011.Sharma B.R., Karki R., Kanneganti T.D. Role of AIM2 inflammasome in inflammatory diseases, cancer and infection. Eur J Immunol. 2019 Nov;49(11):1998-2011.Chen P.A., Shrivastava G., Balcom E.F. et al. Absent in melanoma 2 regulates tumor cell proliferation in glioblastoma multiforme. J Neurooncol. 2019 Sep;144(2):265-273.Chen P.A., Shrivastava G., Balcom E.F. et al. Absent in melanoma 2 regulates tumor cell proliferation in glioblastoma multiforme. J Neurooncol. 2019 Sep;144(2):265-273.Burdette D.L., Russell V.E. STING and the innate immune response to nucleic acids in the cytosol. Nat Immunol. 2013 Jan;14(1):19-26.Burdette D.L., Russell V.E. STING and the innate immune response to nucleic acids in the cytosol. Nat Immunol. 2013 Jan;14(1):19-26.Barber G.N. Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses. Curr Opin Immunol. 2011 Feb;23(1):10-20.Barber G.N. Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses. Curr Opin Immunol. 2011 Feb;23(1):10-20.Ergun S.L., Li L. Structural insights into STING signaling. Trends Cell Biol. 2020 May;30(5):399-407.Ergun S.L., Li L. Structural insights into STING signaling. Trends Cell Biol. 2020 May;30(5):399-407.Xu X.X., Wan H., Nie L. et al. RIG-I: a multifunctional protein beyond a pattern recognition receptor. Protein Cell. 2018 Mar;9(3):246-253.Xu X.X., Wan H., Nie L. et al. RIG-I: a multifunctional protein beyond a pattern recognition receptor. Protein Cell. 2018 Mar;9(3):246-253.Grivennikov S.I., Karin M. Dangerous liaisons: STAT3 and NF-κB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev. 2010 Feb;21(1):11-9.Grivennikov S.I., Karin M. Dangerous liaisons: STAT3 and NF-κB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev. 2010 Feb;21(1):11-9.Cui J., Chen Y., Wang H.Y., Wang R.F. Mechanisms and pathways of innate immune activation and regulation in health and cancer. Hum Vaccin Immunother. 2014;10(11):3270-85.Cui J., Chen Y., Wang H.Y., Wang R.F. Mechanisms and pathways of innate immune activation and regulation in health and cancer. Hum Vaccin Immunother. 2014;10(11):3270-85.

The authors declare that there are no conflicts of interest present.