Identification regulatory noncoding RNAs of human papilloma virus type 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) in cervical tumors
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| 1. | Title | Title of document | Identification regulatory noncoding RNAs of human papilloma virus type 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) in cervical tumors |
| 2. | Creator | Author's name, affiliation, country | Natalia P. Kisseljova; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Maria D. Fedorova; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Anastasia E. Zaikina; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Nadezhda V. Elkina; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Ekaterina E. Goldobina; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Danila S. Elkin; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Pavel M. Abramov; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Larisa S. Pavlova; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 2. | Creator | Author's name, affiliation, country | Svetlana V. Vinokurova; FSBI «National Medical Research Center of Oncology named after N.N. Blokhin» of the Ministry of Health of Russia; Россия |
| 3. | Subject | Discipline(s) | |
| 3. | Subject | Keyword(s) | human papillomaviruses; viral noncoding RNA; cervical cancer |
| 4. | Description | Abstract | 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. |
| 5. | Publisher | Organizing agency, location | Central Research Institute for Epidemiology |
| 6. | Contributor | Sponsor(s) |
RSF grant (15-15-00125) |
| 7. | Date | (DD-MM-YYYY) | 05.09.2022 |
| 8. | Type | Status & genre | Peer-reviewed Article |
| 8. | Type | Type | Research Article |
| 9. | Format | File format | |
| 10. | Identifier | Uniform Resource Identifier | https://virusjour.crie.ru/jour/article/view/602 |
| 10. | Identifier | Digital Object Identifier (DOI) | 10.36233/0507-4088-108 |
| 11. | Source | Title; vol., no. (year) | Problems of Virology; Vol 67, No 3 (2022) |
| 12. | Language | English=en | ru |
| 13. | Relation | Supp. Files |
Fig. 1. Analysis of transcription in the HPV 16 URR by RT-PCR. a: solid line – scheme of the HPV16 URR, located between the late gene L1 and the early genes E6/E7; the positions of the oligonucleotides used for knockdown ncRNAs are marked with asterisks; broken arrows – the start of transcription of early genes [13]; vertical line – the beginning (1 bp) and end (7,904 bp) points of the nucleotide count in the HPV16 circular genome; ORF L1 – the end of the open reading frame of the L1 viral protein; polyA – the signal of polyadenylation of late genes. The black rectangles under solid line – transcripts of early and late genes; gray rectangles – RT-PCR products were detected; white rectangles – RT-PCR products were absent; the numbers inside the rectangles – the positions of forward (F) and reverse (R) primers (bp) according to the HPV16 AF125673 (GenBank) sequence; b–d – electrophoregrams of PCR products: b – SiHa cells; c – CasKi cells; d – cervical tumors. [RT+] and [RT−] – cDNA synthesis was performed in the presence and absence of reverse transcriptase, respectively; [K+] – PCR with HPV16 DNA (positive control for PCR); [K−] – H2O instead of the DNA (control for contamination of PCR reagents); M – 100 bp ladder. (142KB) Fig. 2. Analysis of the transcription direction in the HPV16 URR of SiHa cells by RT-PCR. a: the solid line is a scheme of the HPV16 URR, designations like in legend of Fig. 1 a. F1, F2 and R1, R2 are gene-specific primers for cDNA synthesis on the antisense and sense chains, respectively; b, c – analysis of the products of RT-PCR by electrophoresis in agarose gel. [RT+] and [RT−] – cDNA synthesis was performed in the presence and absence of reverse transcriptase, respectively; [K+] – PCR with DNA of SiHа (positive control for PCR); [K−] – H2O instead of cDNA (control for contamination of PCR reagents); [Pr−] – cDNA synthesis was performed without primers (control for RNA self-priming), adaptor – see «Material and methods»; numbers are the positions of all elements (bp) according to the HPV16 AF125673 (GenBank) sequence; M – marker, 100 bp ladder. The oval indicates the position of the PCR product in the gel. (146KB) Fig. 3. Effect of ncRNA knockdown on the expression and activity of early HPV 16 genes in SiHa cells. a: the solid line is a scheme of the HPV16 early genes region (designations like in legend of Fig. 1); mRNA splicing variants are indicated by a bold line for exons and a thin line for introns, the coding potential of each variant is indicated on the right; the small gray rectangle – the position of examined mRNA region; numbers are the positions of all elements (bp) according to the HPV16 AF125673 (GenBank) sequence; S – transfection with the cocktail of oligonucleotides complementary to the sense chain of ncRNA; AS – transfection with the cocktail of oligonucleotides complementary to the antisense chain of ncRNA; Control – transfection without the addition of oligonucleotides, cDNA synthesized with hexamers; b, c – HPV16 RNA levels normalized regarding to the mRNA of the HPRT gene are indicated on the ordinate axis; b – the mRNA levels of the early genes; c – the levels of ncRNA. Statistical data – a paired t-test (the GraphPad Prism program, v.7.00); d – western blot analysis of p53 expression in cells. А typical transfection result is presented. GAPDH is a control of protein loading; the numbers over the blots indicate normalized p53 protein levels in relation to the control as percentage (calculated by Image Studio Lite Software, v.5.2.). (97KB) Fig. 4. Analysis of the levels of ncRNAs and mRNAs of early HPV16 genes in cervical tumors. The ratio of mRNA copy number to the ncRNA copy number of is indicated on the ordinate axis. The number of copies of each RNA type per 1 ng of initial total RNA was calculated. Black, gray, and white rectangles represent samples with episomal, integrated, and mixed forms of viral DNA, respectively; the shaded rectangle is the SiHa cell line. (66KB) |
| 14. | Coverage | Geo-spatial location, chronological period, research sample (gender, age, etc.) | |
| 15. | Rights | Copyright and permissions |
Copyright (c) 2022 Kisseljova N.P., Fedorova M.D., Zaikina A.E., Elkina N.V., Goldobina E.E., Elkin D.S., Abramov P.M., Pavlova L.S., Vinokurova S.V. This work is licensed under a Creative Commons Attribution 4.0 International License. |