Вопросы вирусологии. 2022; 67: 217-226
Идентификация регуляторных некодирующих РНК вируса папилломы человека типа 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) в опухолях шейки матки
https://doi.org/10.36233/0507-4088-108Аннотация
Введение. Вирусы папилломы человека высокого канцерогенного риска признаны этиологическими агентами рака шейки матки. Постоянная экспрессия вирусных онкобелков Е6 и Е7 необходима для поддержания злокачественного фенотипа опухолевых клеток. Точный механизм регуляции экспрессии вирусных онкогенов в опухолевых клетках до конца не выяснен.
Цель работы – идентификация вирусных некодирующих РНК (нкРНК) при ВПЧ16-положительном раке шейки матки.
Материалы и методы. Для обнаружения вирусных нкРНК в ВПЧ16-положительных первичных плоскоклеточных карциномах шейки матки и клеточных линиях SiHa и CasKi использовали полимеразную цепную реакцию с обратной транскрипцией. Для выяснения функций нкРНК использовали метод нокдауна с олигонуклеотидами, комплементарными нкРНК.
Результаты. Мы идентифицировали нкРНК, транскрибируемые в регуляторной области ВПЧ16, в клеточных линиях и в 32 из 32 плоскоклеточных карцином шейки матки с эписомальной или интегративной формами вирусной ДНК. Нокдаун смысловых или антисмысловых цепей нкРНК приводит к снижению или увеличению уровней мРНК онкогенов Е6 и Е7 в клетках, соответственно. Эти изменения уровней мРНК онкогенов сопровождаются модуляцией уровней белка р53, основной мишени онкобелка Е6.
Заключение. Присутствие впервые выявленных регуляторных нкРНК во всех исследованных опухолях и клеточных линиях свидетельствует об их необходимости для поддержания в них постоянной экспрессии онкогенов Е6 и Е7. Полученные данные могут быть полезны для понимания фундаментальных аспектов регуляции экспрессии вируса в ВПЧ16-позитивных опухолях.
Список литературы
1. Forman D., de Martel C., Lacey C.J., Soerjomataram I., Lortet-Tieulent J., Bruni L., et al. Global burden of human papillomavirus and related. Vaccine. 2012; 30(Suppl. 5): F12–23. https://doi.org/10.1016/j.vaccine.2012.07.055
2. Doorbar J., Egawa N., Griffin H., Kranjec C., Murakami I. Human papillomavirus molecular biology and disease association. Rev. Med. Virol. 2015; 25(Suppl. 1): 2–23. https://doi.org/10.1002/rmv.1822
3. zur Hausen H. Papillomaviruses in the causation of human cancers – a brief historical account. Virology. 2009; 384(2): 260–5. https://doi.org/10.1016/j.virol.2008.11.046
4. Fehrmann F., Laimins L.A. Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation. Oncogene. 2003; 22(33): 5201–7. https://doi.org/10.1038/sj.onc.1206554
5. Goodwin E.C., DiMaio D. Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc. Natl. Acad. Sci. USA. 2000; 97(23): 12513–8. https://doi.org/10.1073/pnas.97.23.12513
6. Goodwin E.C., Yang E., Lee C.J., Lee H. W., DiMaio D., Hwang E.S. Rapid induction of senescence in human cervical carcinoma cells. Proc. Natl. Acad. Sci. USA. 2000; 97(20): 10978–83. https://doi.org/10.1073/pnas.97.20.10978
7. Magaldia T.G., Almsteada L.L., Belloneb S., Prevatt E.G., Santin A.D., DiMaio D. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation. Virology. 2012; 422(1): 114–24. https://doi.org/10.1016/j.virol.2011.10.012
8. Wells S.I., Francis D.A., Karpova A.Y., Dowhanick J.J., Benson J.D., Howley P.M. E2 induces senescence in HPV-positive cells via pRB- and p21CIP-dependent pathways. EMBO J. 2000; 19(21): 762–71. https://doi.org/10.1093/emboj/19.21.5762
9. Prasanth K.V., Spector D.L. Eukaryotic regulatory RNAs: an answer to the ‘genome complexity’ conundrum. Genes Dev. 2007; 21(1): 11–42. https://doi.org/10.1101/gad.1484207
10. Iwakiri D. Multifunctional non-coding Epstein–Barr virus encoded RNAs (EBERs) contribute to viral pathogenesis. Virus Res. 2016; 212: 30–8. https://doi.org/10.1016/j.virusres.2015.08.007
11. Conrad N.K. New insights into the expression and functions of the Kaposi’s sarcoma-associated herpesvirus long noncoding PAN RNA. Virus Res. 2016; 212: 53–63. https://doi.org/10.1016/j.virusres.2015.06.012
12. Klaes R., Woerner S.M., Ridder R., Wentzensen N., Duerst M., Schneider A., et al. Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res. 1999; 59(24): 6132–6.
13. Fedorova M., Vinokurova S., Pavlova L., Komel’kov A., Korolenkova L., Kisseljov F., et al. Human papillomavirus types 16 E1 mRNA is transcribed from P14 early promoter in cervical neoplasms. Virology. 2016; 488: 196–200. https://doi.org/10.1016/j.virol.2015.11.015
14. Szuhai K.V., Bezrookove V., Wiegant J., Vrolijk J., Dirks R.W., Rosenberg C., et al. Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH. Genes Chromosomes Cancer. 2000; 28(1): 92–7. https://doi.org/10.1002/(sici)1098-2264(200005)28:1<92::aid-gcc11>3.0.co;2-2
15. Meissner J.D. Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines. J. Gen. Virol. 1999; 80(Pt. 7): 1725–33. https://doi.org/10.1099/0022-1317-80-7-1725
16. Beiter T., Reich E., Weigert C., Niess A.M., Simon P. Sense or antisense? False priming reverse transcription controls are required for determining sequence orientation by reverse transcription–PCR. Anal. Biochem. 2007; 369(2): 258–61. https://doi.org/10.1016/j.ab.2007.06.044
17. Matsui M., Prakash T.P., Corey D.R. Argonaute 2-dependent regulation of gene expression by single-stranded miRNA mimics. Mol. Ther. 2016; 24(5): 946–55. https://doi.org/10.1038/mt.2016.39
18. Seedorf K., Krämmer G., Dürst M., Suhai S., Röwekamp W.G. Human papillomavirus type 16 DNA sequence. Virology. 1985; 145(1): 181–5. https://doi.org/10.1016/0042-6822(85)90214-4
19. Patrushev L.I., Kovalenko T.F. Functions of noncoding sequences in mammalian genomes. Biochemistry (Mosc.). 2014; 79(13): 1442–69. https://doi.org/10.1134/S0006297914130021
20. Cripe T.P., Haugen T.H., Turket J.P., Tabatabai F., Schmid P.G. 3rd, Dürst M., et al. Transcriptional regulation of the human papillomavirus- 16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis. EMBO J. 1987; 6(12): 3745–53. https://doi.org/10.1002/j.1460-2075.1987.tb02709.x
21. Melgar M.F., Collins F.S., Sethupathy P. Discovery of active enhancers through bidirectional expression of short transcripts. Genome Biol. 2011; 12(11): R113. https://doi.org/10.1186/gb-2011-12-11-r113
22. Scheffner M., Huibregtse J.M., Vierstra R.D., Howley P.M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993; 75(3): 495–505. https://doi.org/10.1016/0092-8674(93)90384-3
23. Melsheimer P., Vinokurova S., Wentzensen N., Bastert G., von Knebel Doeberitz M. DNA aneuploidy and integration of human papillomavirus type 16 E6/E7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri. Clin. Cancer Res. 2004; 10(9): 3059–63. https://doi.org/10.1158/1078-0432.ccr-03-0565
24. Kassab M.A., Mudassir M., Singh A., Muthuraman N., Bhagat M., Palanichamy J.K., et al. Gene silencing and activation of human papillomavirus 18 is modulated by sense promoter associated RNA in bidirectionally transcribed long control region. PLoS One. 2015; 10(6): e0128416. https://doi.org/10.1371/journal.pone.0128416
25. Han J., Kim D., Morris K.V. Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. Proc. Natl. Acad. Sci. USA. 2007; 104(30): 12422–7. https://doi.org/10.1073/pnas.0701635104
Problems of Virology. 2022; 67: 217-226
Identification regulatory noncoding RNAs of human papilloma virus type 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) in cervical tumors
https://doi.org/10.36233/0507-4088-108Abstract
Introduction. High carcinogenic-risk human papillomaviruses (hrHPVs) are recognized as etiological agents of cervical cancer. Constant expression of the viral oncoproteins, E6 and E7, is required for maintenance of the malignant phenotype of tumor cells. The exact mechanism of regulation of viral oncogenes expression in tumor cells is not fully elucidated.
The purpose: identification of viral noncoding RNAs (ncRNAs) in HPV16-positve cervical cancer.
Materials and methods. The reverse transcription polymerase chain reactions were used to detect viral ncRNAs in HPV16-positve primary cervical squamous cell carcinomas and SiHa and CasKi cell lines. The knockdown technique with oligonucleotides complementary to ncRNAs was used to elucidate their functions.
Results. We have identified ncRNAs transcribed in the upstream regulatory region of HPV16 in the cervical carcinoma cell lines and in 32 out 32 cervical squamous cell carcinomas with episomal or integrated forms of HPV16 DNA. Knockdown of sense or antisense strains of ncRNAs by oligonucleotides results in a decrease or increase of the E6 and E7 oncogenes mRNA levels in cells, respectively. These changes of oncogenes mRNA levels are accompanied by the modulation of the levels of the p53 protein, the main target of the E6 oncoprotein.
Conclusion. The presence of regulatory ncRNAs in all examined tumors and cell lines revealed for the first time indicates their necessity for maintenance of constant expression of E6 and E7 oncogenes in them. The findings can be useful for understanding of the fundamental aspects of the viral expression regulation in HPV16-positive tumors.
References
1. Forman D., de Martel C., Lacey C.J., Soerjomataram I., Lortet-Tieulent J., Bruni L., et al. Global burden of human papillomavirus and related. Vaccine. 2012; 30(Suppl. 5): F12–23. https://doi.org/10.1016/j.vaccine.2012.07.055
2. Doorbar J., Egawa N., Griffin H., Kranjec C., Murakami I. Human papillomavirus molecular biology and disease association. Rev. Med. Virol. 2015; 25(Suppl. 1): 2–23. https://doi.org/10.1002/rmv.1822
3. zur Hausen H. Papillomaviruses in the causation of human cancers – a brief historical account. Virology. 2009; 384(2): 260–5. https://doi.org/10.1016/j.virol.2008.11.046
4. Fehrmann F., Laimins L.A. Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation. Oncogene. 2003; 22(33): 5201–7. https://doi.org/10.1038/sj.onc.1206554
5. Goodwin E.C., DiMaio D. Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc. Natl. Acad. Sci. USA. 2000; 97(23): 12513–8. https://doi.org/10.1073/pnas.97.23.12513
6. Goodwin E.C., Yang E., Lee C.J., Lee H. W., DiMaio D., Hwang E.S. Rapid induction of senescence in human cervical carcinoma cells. Proc. Natl. Acad. Sci. USA. 2000; 97(20): 10978–83. https://doi.org/10.1073/pnas.97.20.10978
7. Magaldia T.G., Almsteada L.L., Belloneb S., Prevatt E.G., Santin A.D., DiMaio D. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation. Virology. 2012; 422(1): 114–24. https://doi.org/10.1016/j.virol.2011.10.012
8. Wells S.I., Francis D.A., Karpova A.Y., Dowhanick J.J., Benson J.D., Howley P.M. E2 induces senescence in HPV-positive cells via pRB- and p21CIP-dependent pathways. EMBO J. 2000; 19(21): 762–71. https://doi.org/10.1093/emboj/19.21.5762
9. Prasanth K.V., Spector D.L. Eukaryotic regulatory RNAs: an answer to the ‘genome complexity’ conundrum. Genes Dev. 2007; 21(1): 11–42. https://doi.org/10.1101/gad.1484207
10. Iwakiri D. Multifunctional non-coding Epstein–Barr virus encoded RNAs (EBERs) contribute to viral pathogenesis. Virus Res. 2016; 212: 30–8. https://doi.org/10.1016/j.virusres.2015.08.007
11. Conrad N.K. New insights into the expression and functions of the Kaposi’s sarcoma-associated herpesvirus long noncoding PAN RNA. Virus Res. 2016; 212: 53–63. https://doi.org/10.1016/j.virusres.2015.06.012
12. Klaes R., Woerner S.M., Ridder R., Wentzensen N., Duerst M., Schneider A., et al. Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes. Cancer Res. 1999; 59(24): 6132–6.
13. Fedorova M., Vinokurova S., Pavlova L., Komel’kov A., Korolenkova L., Kisseljov F., et al. Human papillomavirus types 16 E1 mRNA is transcribed from P14 early promoter in cervical neoplasms. Virology. 2016; 488: 196–200. https://doi.org/10.1016/j.virol.2015.11.015
14. Szuhai K.V., Bezrookove V., Wiegant J., Vrolijk J., Dirks R.W., Rosenberg C., et al. Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH. Genes Chromosomes Cancer. 2000; 28(1): 92–7. https://doi.org/10.1002/(sici)1098-2264(200005)28:1<92::aid-gcc11>3.0.co;2-2
15. Meissner J.D. Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines. J. Gen. Virol. 1999; 80(Pt. 7): 1725–33. https://doi.org/10.1099/0022-1317-80-7-1725
16. Beiter T., Reich E., Weigert C., Niess A.M., Simon P. Sense or antisense? False priming reverse transcription controls are required for determining sequence orientation by reverse transcription–PCR. Anal. Biochem. 2007; 369(2): 258–61. https://doi.org/10.1016/j.ab.2007.06.044
17. Matsui M., Prakash T.P., Corey D.R. Argonaute 2-dependent regulation of gene expression by single-stranded miRNA mimics. Mol. Ther. 2016; 24(5): 946–55. https://doi.org/10.1038/mt.2016.39
18. Seedorf K., Krämmer G., Dürst M., Suhai S., Röwekamp W.G. Human papillomavirus type 16 DNA sequence. Virology. 1985; 145(1): 181–5. https://doi.org/10.1016/0042-6822(85)90214-4
19. Patrushev L.I., Kovalenko T.F. Functions of noncoding sequences in mammalian genomes. Biochemistry (Mosc.). 2014; 79(13): 1442–69. https://doi.org/10.1134/S0006297914130021
20. Cripe T.P., Haugen T.H., Turket J.P., Tabatabai F., Schmid P.G. 3rd, Dürst M., et al. Transcriptional regulation of the human papillomavirus- 16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis. EMBO J. 1987; 6(12): 3745–53. https://doi.org/10.1002/j.1460-2075.1987.tb02709.x
21. Melgar M.F., Collins F.S., Sethupathy P. Discovery of active enhancers through bidirectional expression of short transcripts. Genome Biol. 2011; 12(11): R113. https://doi.org/10.1186/gb-2011-12-11-r113
22. Scheffner M., Huibregtse J.M., Vierstra R.D., Howley P.M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993; 75(3): 495–505. https://doi.org/10.1016/0092-8674(93)90384-3
23. Melsheimer P., Vinokurova S., Wentzensen N., Bastert G., von Knebel Doeberitz M. DNA aneuploidy and integration of human papillomavirus type 16 E6/E7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri. Clin. Cancer Res. 2004; 10(9): 3059–63. https://doi.org/10.1158/1078-0432.ccr-03-0565
24. Kassab M.A., Mudassir M., Singh A., Muthuraman N., Bhagat M., Palanichamy J.K., et al. Gene silencing and activation of human papillomavirus 18 is modulated by sense promoter associated RNA in bidirectionally transcribed long control region. PLoS One. 2015; 10(6): e0128416. https://doi.org/10.1371/journal.pone.0128416
25. Han J., Kim D., Morris K.V. Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. Proc. Natl. Acad. Sci. USA. 2007; 104(30): 12422–7. https://doi.org/10.1073/pnas.0701635104
