Russian Journal of BiotherapyRussian Journal of BiotherapyРоссийский биотерапевтический журнал1726-97841726-9792Publishing House ABV Press114310.17650/1726-9784-2019-18-1-67-74ORIGINAL REPORTSОРИГИНАЛЬНЫЕ СТАТЬИBRCA1 participates in the expression of noncoding XIST RNABRCA1 участвует в экспрессии некодирующей РНК XISThttps://orcid.org/0000-0002-0158-4605ShestakovaE. A.ШестаковаЕ. А.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

elenaanshestakova@mail.ruhttps://orcid.org/0000-0002-7673-4284BogushT. A.БогушТ. А.
Russian Federation

24 Kashirskoe Shosse, Moscow 115478

115478 Москва, Каширское шоссе, 24

N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian FederationФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава России1904201918167741904201919042019https://bioterapevt.abvpress.ru/jour/article/view/1143

Introduction . Noncoding RNA of XIST gene (X inactivation-specific transcript) initiates inactivation of one of X chromosomes in cells of female organism. Further stages of this process include chromatin epigenetic modifications leading to the inhibition of the most genes on X chromosome. Recently the data were obtained that tumor suppressor BRCA1 is associated with inactive X chromosome (Xi) participating in XIST RNA localization on Xi and influencing XIST RNA expression.Objective: to reveal the role of BRCA1 in XIST RNA expression.Materials and methods . The objects of the study were mutant breast cancer cell lines (BRCA1–/–): HCC1395, HCC1937, SUM149PT, and, as controls – cell lines containing wild type of BRCA1 gene (BRCA1+/+): IMR90 и 293T. Method of reverse transcription coupled with polymerase chain reaction (RT-PCR) was used for the analysis of XIST RNA expression.Results . In the clone of doxycycline-inducible HCC1937 breast cancer cell line XIST RNA expression was observed upon BRCA1 induction. In HCC1395, HCC1937 and SUM149PT breast cancer cell lines containing mutant BRCA1 gene (BRCA1–/–) and nonfunctional BRCA1 protein the absence of XIST RNA expression was observed using RT-PCR. This observation indicates the indispensable role of functional BRCA1 protein in XIST RNA expression.Conclusion . Altogether, the data obtained in this study confirm the role of BRCA1 in the expression of noncoding inhibiting XIST RNA and suggest the involvement of BRCA1 in the inhibition of gene expression on Xi.

Введение . Некодирующая РНК гена XIST (X inactivation-specific transcript) инициирует процесс инактивации одной из хромосом Х в клетках женского организма. Последующие стадии этого процесса включают эпигенетические модификации хроматина, что приводит к ингибированию экспрессии большинства генов на Х-хромосоме. В последние годы получены данные, свидетельствующие о том, что белок – супрессор опухолей BRCA1 взаимодействует с неактивной Х-хромосомой (inactive X chromosome, Xi), влияя на локализацию и транскрипцию РНК XIST.Цель исследования – изучение роли BRCA1 в индукции экспрессии РНК XIST.Материалы и методы . Объектом исследования служили клеточные линии рака молочной железы (РМЖ), мутантные по гену BRCA1 (BRCA1–/–): HCC1395, HCC1937, SUM149PT, и в качестве контроля – клеточные линии, содержащие ген BRCA1 дикого типа (BRCA1+/+): IMR90 и 293T. Для анализа экспрессии РНК XIST использовали полимеразную цепную реакцию с обратной транскрипцией (ОТ-ПЦР).Результаты . В клоне доксициклин-индуцированной клеточной линии РМЖ HCC1937 наблюдалась экспрессия РНК XIST при индукции BRCA1 (инициации экспрессии гена BRCA1 с последующим синтезом белка BRCA1, который активирует экспрессию РНК XIST, т. е. ее транскрипцию с гена XIST). В клеточных линиях РМЖ HCC1395, HCC1937, SUM149PT, мутантных по гену BRCA1 (BRCA1–/–) и содержащих нефункциональный белок BRCA1, с помощью ОТ-ПЦР экспрессии РНК XIST не обнаружено. Это говорит о важной роли функционального белка BRCA1 в индукции экспрессии данной РНК. Заключение . Полученные данные свидетельствуют о роли BRCA1 в экспрессии некодирующей инактивирующей РНК XIST и указывают на участие BRCA1 в ингибировании экспрессии генов на Xi.

breast cancercell linesBRCA1XIST RNA expressionрак молочной железыклеточные линииBRCA1экспрессия РНК XIST1. Galupa R., Heard E. X-chromosome inactivation: new insights into cis and trans regulation. Curr Opin Genet Dev 2015;31:57–66. DOI: 10.1016/j.gde.2015.04.002. PMID: 26004255.Galupa R., Heard E. X-chromosome inactivation: new insights into cis and trans regulation. Curr Opin Genet Dev 2015;31:57–66. DOI: 10.1016/j.gde.2015.04.002. PMID: 26004255.2. Heard E. Recent advances in X-chromosome inactivation. Curr Opin Cell Biol 2004;16(3):247–55. DOI: 10.1016/j.ceb.2004.03.005. PMID: 15145348.Heard E. Recent advances in X-chromosome inactivation. Curr Opin Cell Biol 2004;16(3):247–55. DOI: 10.1016/j.ceb.2004.03.005. PMID: 15145348.3. Chadwick B.P., Willard H.F. Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome. Proc Natl Acad Sci USA 2004;101(50):17450–5. DOI: 10.1073/pnas.0408021101. PMID: 15574503.Chadwick B.P., Willard H.F. Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome. Proc Natl Acad Sci USA 2004;101(50):17450–5. DOI: 10.1073/pnas.0408021101. PMID: 15574503.4. Chadwick B.P., Lane T.F. BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation. Chromosoma 2005;114(6):432–9. DOI: 10.1007/s00412-005-0029-1. PMID: 16240122.Chadwick B.P., Lane T.F. BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation. Chromosoma 2005;114(6):432–9. DOI: 10.1007/s00412-005-0029-1. PMID: 16240122.5. Pageau G.J., Lawrence J.B. BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin. J Cell Biol 2006;175(5):693–701. DOI: 10.1083/jcb.200602055. PMID: 17145961.Pageau G.J., Lawrence J.B. BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin. J Cell Biol 2006;175(5):693–701. DOI: 10.1083/jcb.200602055. PMID: 17145961.6. Ganesan S.C., Silver D.P., Greenberg R.A. et al. BRCA1 supports XIST RNA concentration on the inactive X chromosome. Cell 2002;111(3):393–405. PMID: 12419249.Ganesan S.C., Silver D.P., Greenberg R.A. et al. BRCA1 supports XIST RNA concentration on the inactive X chromosome. Cell 2002;111(3):393–405. PMID: 12419249.7. Silver D.P., Dimitrov S.D., Feunteun J. et al. Further evidence for BRCA1 communication with the inactive X chromosome. Cell 2007;128(5): 991–1002. DOI: 10.1016/j. cell.2007.02.025. PMID: 17350581.Silver D.P., Dimitrov S.D., Feunteun J. et al. Further evidence for BRCA1 communication with the inactive X chromosome. Cell 2007;128(5): 991–1002. DOI: 10.1016/j. cell.2007.02.025. PMID: 17350581.8. Welcsh P.L., Lee M.K., GonzalezHernandez R.M. et al. BRCA1 transcriptionally regulates genes involved in breast tumorigenesis. Proc Natl Acad Sci USA 2002;99(11):7560–5. DOI: 10.1073/pnas.062181799. PMID: 12032322.Welcsh P.L., Lee M.K., GonzalezHernandez R.M. et al. BRCA1 transcriptionally regulates genes involved in breast tumorigenesis. Proc Natl Acad Sci USA 2002;99(11):7560–5. DOI: 10.1073/pnas.062181799. PMID: 12032322.9. Shestakova E.A. Epigenetic regulation of BRCA1 expression and its role in breast cancer stem cell development. Turk J Biol 2016;40(5):981–9. DOI: 10.3906/biy-1507-145.Shestakova E.A. Epigenetic regulation of BRCA1 expression and its role in breast cancer stem cell development. Turk J Biol 2016;40(5):981–9. DOI: 10.3906/biy-1507-145.10. Богуш Т.А., Шестакова Е.А., Вихлянцева Н.О. и др. Эпигенетические механизмы регуляции BRCA1. Онкогинекология 2017;23(2):4–11. [Bogush T.A., Shestakova E.A., Vikhlyantseva N.О. et al. Epigenetic mechanisms of regulation of BRCA1. Onkoginekologiya = Oncogynecology 2017;23(2):4–11. (In Russ.)].Богуш Т.А., Шестакова Е.А., Вихлянцева Н.О. и др. Эпигенетические механизмы регуляции BRCA1. Онкогинекология 2017;23(2):4–11. [Bogush T.A., Shestakova E.A., Vikhlyantseva N.О. et al. Epigenetic mechanisms of regulation of BRCA1. Onkoginekologiya = Oncogynecology 2017;23(2):4–11. (In Russ.)].11. Elstrodt F., Hollestelle A., Nagel J.H. et al. BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. Cancer Res 2006;66(1):41–5. DOI: 10.1158/0008-5472.CAN-05-2853. PMID: 16397213.Elstrodt F., Hollestelle A., Nagel J.H. et al. BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. Cancer Res 2006;66(1):41–5. DOI: 10.1158/0008-5472.CAN-05-2853. PMID: 16397213.12. Karami F., Mehdipour P. A comprehensive focus on global spectrum of BRCA1 and BRCA2 mutations in breast cancer. Biomed Res Int 2013;2013: 928562. DOI: 10.1155/2013/928562. PMID: 24312913.Karami F., Mehdipour P. A comprehensive focus on global spectrum of BRCA1 and BRCA2 mutations in breast cancer. Biomed Res Int 2013;2013: 928562. DOI: 10.1155/2013/928562. PMID: 24312913.13. Wang F., Fang Q., Ge Z. et al. Common BRCA1 and BRCA2 mutations in breast cancer families: a meta-analysis from systematic review. Mol Biol Rep 2012;39(3):2109–18. DOI: 10.1007/ s11033-011-0958-0. PMID: 21643751.Wang F., Fang Q., Ge Z. et al. Common BRCA1 and BRCA2 mutations in breast cancer families: a meta-analysis from systematic review. Mol Biol Rep 2012;39(3):2109–18. DOI: 10.1007/ s11033-011-0958-0. PMID: 21643751.14. Iyevleva A.G., Suspitsin E.N., Kroeze K. et al. Non-founder BRCA1 mutations in Russian breast cancer patients. Cancer Lett 2010;298(2):258–63. DOI: 10.1016/j.canlet.2010.07.013. PMID: 20727672.Iyevleva A.G., Suspitsin E.N., Kroeze K. et al. Non-founder BRCA1 mutations in Russian breast cancer patients. Cancer Lett 2010;298(2):258–63. DOI: 10.1016/j.canlet.2010.07.013. PMID: 20727672.15. Stadler Z.K., Salo-Mullen E., Patil S.M. et al. Prevalence of BRCA1 and BRCA2 mutations in Ashkenazi Jewish families with breast and pancreatic cancer. Cancer 2012;118(2):493–9. DOI: 10.1002/cncr.26191. PMID: 21598239.Stadler Z.K., Salo-Mullen E., Patil S.M. et al. Prevalence of BRCA1 and BRCA2 mutations in Ashkenazi Jewish families with breast and pancreatic cancer. Cancer 2012;118(2):493–9. DOI: 10.1002/cncr.26191. PMID: 21598239.16. Stephens P.J., McBride D.J., Lin M.L. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 2009;462(7276):1005– 10. DOI: 10.1038/nature08645. PMID: 20033038.Stephens P.J., McBride D.J., Lin M.L. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 2009;462(7276):1005– 10. DOI: 10.1038/nature08645. PMID: 20033038.17. Olsson E., Winter C., George A. et al. Mutation screening of 1,237 cancer genes across Six model cell lines of basal-like breast cancer. PLoS One 2015;10(12):e0144528. DOI: 10.1371/journal.pone.0144528. PMID: 26670335.Olsson E., Winter C., George A. et al. Mutation screening of 1,237 cancer genes across Six model cell lines of basal-like breast cancer. PLoS One 2015;10(12):e0144528. DOI: 10.1371/journal.pone.0144528. PMID: 26670335.18. Lehmann B.D., Bauer J.A., Chen X. et al. Identification of human triplenegative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011;121(7):2750–67. DOI: 10.1172/JCI45014. PMID: 21633166.Lehmann B.D., Bauer J.A., Chen X. et al. Identification of human triplenegative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011;121(7):2750–67. DOI: 10.1172/JCI45014. PMID: 21633166.19. Robert-Fortel I., Junera H.R., Geraud G. et al. Three-dimentional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy. Chromosoma 1993;102(3):146–57. PMID: 7681367.Robert-Fortel I., Junera H.R., Geraud G. et al. Three-dimentional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy. Chromosoma 1993;102(3):146–57. PMID: 7681367.20. Pageau G.J., Hall L.L., Lawrence J.B. BRCA1 does not paint the inactive X to localize XIST RNA but may contribute to broad changes in cancer that impact XIST and Xi heterochromatin. J Cell Biochem 2007;100(4):835–50. DOI: 10.1002/jcb.21188. PMID: 17146760.Pageau G.J., Hall L.L., Lawrence J.B. BRCA1 does not paint the inactive X to localize XIST RNA but may contribute to broad changes in cancer that impact XIST and Xi heterochromatin. J Cell Biochem 2007;100(4):835–50. DOI: 10.1002/jcb.21188. PMID: 17146760.21. Sirchia S.M., Tabano S., Monti L. et al. Misbehaviour of XIST RNA in breast cancer cells. PLoS One 2009;4(5):e5559. DOI: 10.1371/journal.pone.0005559. PMID: 19440381.Sirchia S.M., Tabano S., Monti L. et al. Misbehaviour of XIST RNA in breast cancer cells. PLoS One 2009;4(5):e5559. DOI: 10.1371/journal.pone.0005559. PMID: 19440381.22. Richardson A.L., Wang Z.C., De Nicolo A. et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 2006;9(2):121–32. DOI: 10.1016/j.ccr.2006.01.013. PMID: 16473279.Richardson A.L., Wang Z.C., De Nicolo A. et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 2006;9(2):121–32. DOI: 10.1016/j.ccr.2006.01.013. PMID: 16473279.23. Поспехова Н.И. Комплексный анализ наследственной формы рака молочной железы и/или яичников: молекулярно-генетические и фенотипические характеристики. Автореф. дис. ... д-ра мед. наук. М., 2011. [Pospekhova N.I. Complex analysis of hereditary form of breast and/or ovarian cancer: molecular genetic and phenotypic characteristics. Author’s abstract of thesis ... of doctor of medical sciences. Moscow, 2011. (In Russ.)].Поспехова Н.И. Комплексный анализ наследственной формы рака молочной железы и/или яичников: молекулярно-генетические и фенотипические характеристики. Автореф. дис. ... д-ра мед. наук. М., 2011. [Pospekhova N.I. Complex analysis of hereditary form of breast and/or ovarian cancer: molecular genetic and phenotypic characteristics. Author’s abstract of thesis ... of doctor of medical sciences. Moscow, 2011. (In Russ.)].