Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma
Abstract
:1. Introduction
2. Materials and Methods
2.1. Subjects
2.2. Detection of Human Papillomavirus (HPV) DNA and Quantitative Polymerase Chain Reaction (PCR) Analysis of HPV-6 Viral Load
2.2.1. Extraction of Genomic DNA from Laryngeal Papilloma (LP) and PCR Conditions
2.2.2. Partial Cloning of the HPV-6-LCR Region and Identification of HPV-6 Subtypes
2.2.3. Quantitative PCR Analysis of HPV-6 Viral Load in LP
2.3. Measurement of Viral mRNA Expression in HPV-6-infected LP by Quantitative Real-Time PCR
2.4. In Situ Hybridization (ISH) with HPV DNA Probes
2.5. RNA-ISH with HPV-6 E6, E2, E4, E5a, and E5b Digoxigenin RNA Probes
2.6. Double Fluorescence RNA-ISH
2.7. Immunohistochemistry for HPV-6 E4
2.7.1. Preparation of an Anti-HPV-6 E4 Monoclonal Antibody
2.7.2. Evaluation of Antibody Specificity
2.7.3. Immunohistochemistry for HPV-6 E1^E4
2.8. Statistical Analysis
2.9. Ethics Approval and Consent to Participate
3. Results
3.1. HPV DNA Distribution, Subtypes, and Viral Load in LP
3.2. Expression of HPV-6 mRNAs in LP
3.2.1. Expression Levels of the 9 Viral mRNAs
3.2.2. Viral Load Alteration and Levels of the 9 Viral mRNAs in LP during 80 Months
3.2.3. HPV-6 E6, E2, E4, E5a, and E5b mRNA Expression in LP Detected by RNA-ISH
3.3. Immunohistochemistry for HPV-6 E1^E4
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Case | Age (Years) | Sex | Surgery | No. of Tumors | Subsite | Derkay Score | HPV Subtype |
---|---|---|---|---|---|---|---|
1 | 27 | M | 3rd | Multiple | B, TVC; B, FVC | 15 | HPV-6a |
2 | 43 | M | 1st | multiple | B, TVC; L, FVC | 13 | HPV-6b |
3 | 34 | M | 2nd | multiple | B, FVC | 6 | HPV-6b |
4 | 41 | M | 1st | multiple | B, TVC | 4 | HPV-6vc |
5 | 41 | M | 1st | single | R, TVC | 2 | HPV-6vc |
6 | 17 | F | 1st | multiple | B, TVC | 6 | HPV-6vc |
7 | 28 | M | 1st | multiple | B, TVC | 2 | HPV6-a |
8 | 4 | F | 1st | multiple | B, TVC | 6 | HPV-6vc |
9 | 59 | M | 1st | multiple | B, TVC | 6 | HPV6-vc |
61 | 4th | multiple | B, TVC | 3 | |||
10 | 67 | M | 1st | multiple | B, TVC | 2 | HPV6-vc |
68 | 2nd | multiple | B, TVC | 14 | |||
69 | 3rd | multiple | L, TVC | 17 | |||
69 | 4th | multiple | B, TVC | 12 | |||
71 | 5th | multiple | B, TVC | 6 | |||
72 | 6th | multiple | B, TVC | 11 | |||
73 | 7th | multiple | B, TVC | 14 | |||
73 | 8th | multiple | B, TVC | 15 | |||
73 | 9th | multiple | B, TVC | 12 | |||
74 | 10th | multiple | B, TVC | 15 | |||
11 | 45 | M | 1st | multiple | B, TVC | 6 | HPV-6 |
12 | 44 | M | 1st | single | R, TVC | 3 | HPV-6a |
13 | 44 | M | 1st | single | L, TVC | 2 | HPV-6 |
Case | Surgery | Viral Load (Copies/ng DNA) | HPV mRNA Expression/β-actin | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
E6 | E7 | E1 | E2 | E4 | E5a | E5b | L2 | L1 | |||
1 | 3rd | 2,268,452 | 0.110 | 0.179 | 0.026 | 0.185 | 10.675 | 4.766 | 2.900 | 0.010 | 0.012 |
2 | 1st | 649,319 | 0.335 | 0.312 | 0.105 | 0.159 | 4.496 | 24.633 | 8.496 | 0.062 | 0.134 |
3 | 2nd | 436 | 0.134 | 0.163 | 0.010 | 0.109 | 0.677 | 2.585 | 1.513 | 0.002 | 0.006 |
4 | 1st | 21,372 | 0.167 | 0.317 | 0.000 | 0.317 | 14.643 | 1.524 | 11.429 | 0.000 | 0.017 |
5 | 1st | 25,347 | 0.425 | 0.153 | 0.010 | 0.213 | 11.474 | 2.241 | 2.309 | 0.001 | 0.002 |
6 | 1st | 4739 | 0.135 | 0.218 | 0.003 | 0.059 | 16.934 | 0.004 | 4.460 | 0.000 | 0.010 |
7 | 1st | 14,170 | 0.032 | 0.100 | 0.006 | 0.099 | 6.175 | 1.068 | 1.035 | 0.000 | 0.001 |
8 | 1st | 36,408 | 0.394 | 0.554 | 0.018 | 0.322 | 11.688 | 0.278 | 6.403 | 0.002 | 0.004 |
9 | 1st | 2,199,081 | 0.587 | 0.208 | 0.019 | 0.122 | 18.409 | 1.108 | 4.032 | 0.002 | 0.008 |
4th | 2,918,599 | 0.542 | 2.799 | 0.693 | 5.824 | 20,828.885 | 74.591 | 256.091 | 6.180 | 0.775 | |
10 | 1st | 166,859 | 0.501 | 1.627 | 0.991 | 2.838 | 149.595 | 16.359 | 32.002 | 0.118 | 0.048 |
2nd | 17,173 | 0.634 | 1.841 | 0.448 | 1.806 | 42.551 | 4.378 | 12.183 | 0.025 | 0.020 | |
3rd | 39,002 | 0.401 | 0.177 | 0.027 | 0.223 | 18.655 | 3.723 | 3.061 | 0.002 | 0.007 | |
4th | 28,595 | NA | NA | NA | NA | NA | NA | NA | NA | NA | |
5th | 153,164 | 0.500 | 1.543 | 0.790 | 2.312 | 165.797 | 14.312 | 25.824 | 0.318 | 0.283 | |
6th | 9551 | 0.083 | 0.092 | 0.007 | 0.103 | 5.951 | 1.657 | 0.796 | 0.001 | 0.001 | |
7th | 645,354 | 0.566 | 1.893 | 0.946 | 2.516 | 231.081 | 26.454 | 51.181 | 0.190 | 0.423 | |
8th | 24,404 | 0.849 | 2.830 | 1.132 | 3.716 | 290.630 | 23.532 | 52.166 | 0.373 | 0.477 | |
9th | 6078 | 0.745 | 1.858 | 0.298 | 1.524 | 44.088 | 4.409 | 7.366 | 0.027 | 0.011 | |
10th | 838,013 | 0.406 | 0.960 | 0.269 | 0.947 | 33.520 | 3.924 | 6.529 | 0.040 | 0.009 | |
11 | 1st | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
12 | 1st | 19,850 | NA | NA | NA | NA | NA | NA | NA | NA | NA |
13 | 1st | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
Case | DNA-ISH | HPV RNA-ISH | E4-IHC | ||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
E6 | E2 | E4 | E5a | E5b | |||||||||||||||||
L | M | U | L | M | U | L | M | U | L | M | U | L | M | U | L | M | U | L | M | U | |
1 * | − | 1+ | 1+ | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
2 | 1+ | 1+ | 3+ | − | − | − | − | − | − | − | − | 1+ | − | 2+ | 3+ | − | − | − | − | 1+ | 2+ |
3 | 3+ | 3+ | 3+ | − | − | 1+ | − | 1+ | − | − | 1+ | 1+ | − | 1+ | 2+ | − | 1+ | 2+ | − | − | 1+ |
4 | 3+ | 3+ | 3+ | − | − | − | − | − | 1+ | − | − | 1+ | − | − | − | − | − | − | − | 1+ | 1+ |
5 | 3+ | 3+ | 3+ | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | 1+ | 1+ |
6 | − | 2+ | 2+ | − | − | − | − | − | 1+ | − | − | 2+ | − | − | 1+ | − | − | − | − | 2+ | 2+ |
7 | 2+ | 2+ | 2+ | − | − | − | − | − | − | − | − | 2+ | − | − | − | − | − | − | − | 1+ | 2+ |
8 | 1+ | 3+ | 2+ | − | − | − | − | − | − | − | 1+ | 1+ | − | − | 1+ | − | − | − | − | 1+ | 1+ |
9 | − | 3+ | 3+ | − | − | 1+ | − | − | 1+ | − | 2+ | 3+ | − | 1+ | 2+ | − | − | 1+ | − | 2+ | 3+ |
10 | − | 3+ | 3+ | − | − | 1+ | − | − | − | − | 1+ | 2+ | − | 1+ | 2+ | − | 1+ | 2+ | − | 2+ | 3+ |
11 | 1+ | 2+ | 3+ | − | 2+ | 2+ | 2+ | 3+ | 2+ | − | 2+ | 3+ | − | 1+ | 1+ | − | 2+ | 3+ | − | 2+ | 3+ |
12 | 1+ | 1+ | 3+ | − | − | − | − | − | 1+ | − | − | 2+ | − | 2+ | 3+ | − | − | 1+ | − | 1+ | 2+ |
13 | 2+ | 3+ | 3+ | − | 1+ | 2+ | − | − | − | − | 1+ | 2+ | − | 1+ | 1+ | − | 1+ | 1+ | − | 1+ | 2+ |
E6 | E7 | E1 | E2 | E4 | E5a | E5b | L2 | L1 | |
---|---|---|---|---|---|---|---|---|---|
E6 | - | 0.786 *** | 0.757 *** | 0.754 *** | 0.807 *** | 0.498 * | 0.705 *** | 0.650 *** | 0.577 *** |
E7 | 0.786 *** | - | 0.794 *** | 0.882 *** | 0.858 *** | 0.649 ** | 0.942 *** | 0.789 *** | 0.838 *** |
E1 | 0.757 *** | 0.794 *** | - | 0.853 *** | 0.780 *** | 0.845 *** | 0.766 *** | 0.944 *** | 0.745 *** |
E2 | 0.754 *** | 0.882 *** | 0.853 *** | - | 0.861 *** | 0.721 *** | 0.872 *** | 0.819 *** | 0.743 *** |
E4 | 0.807 *** | 0.858 *** | 0.780 *** | 0.861 *** | - | 0.554 *** | 0.828 *** | 0.705 *** | 0.710 *** |
E5a | 0.498 * | 0.649 ** | 0.845 *** | 0.721 *** | 0.554 *** | - | 0.6965 *** | 0.904 *** | 0.814 *** |
E5b | 0.705 *** | 0.942 *** | 0.766 *** | 0.872 *** | 0.828 *** | 0.6965 *** | - | 0.781 *** | 0.922 *** |
L2 | 0.650 *** | 0.789 *** | 0.944 *** | 0.819 *** | 0.705 *** | 0.904 *** | 0.781 *** | - | 0.806 *** |
L1 | 0.577 *** | 0.838 *** | 0.745 *** | 0.743 *** | 0.710 *** | 0.814 *** | 0.922 *** | 0.806 *** | - |
mRNA | r | p-Value |
---|---|---|
E6 | 0.237 | 0.3289 |
E7 | 0.277 | 0.2506 |
E1 | 0.449 | 0.0536 |
E2 | 0.395 | 0.0944 |
E4 | 0.323 | 0.1777 |
E5a | 0.507 | 0.0267 * |
E5b | 0.374 | 0.1150 |
L2 | 0.542 | 0.0165 * |
L1 | 0.436 | 0.0619 |
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Ikegami, T.; Hirakawa, H.; Tsukahara, N.; Murakami, A.; Kise, N.; Kiyuna, A.; Kosugi, T.; Agena, S.; Kinjyo, H.; Hasegawa, N.; Touyama, M.; Kondo, S.; Maeda, H.; Suzuki, M.; Ganaha, A. Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma. Microorganisms 2021, 9, 520. https://doi.org/10.3390/microorganisms9030520
Ikegami T, Hirakawa H, Tsukahara N, Murakami A, Kise N, Kiyuna A, Kosugi T, Agena S, Kinjyo H, Hasegawa N, Touyama M, Kondo S, Maeda H, Suzuki M, Ganaha A. Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma. Microorganisms. 2021; 9(3):520. https://doi.org/10.3390/microorganisms9030520
Chicago/Turabian StyleIkegami, Taro, Hitoshi Hirakawa, Narutoshi Tsukahara, Akikazu Murakami, Norimoto Kise, Asanori Kiyuna, Takayoshi Kosugi, Shinya Agena, Hidetoshi Kinjyo, Narumi Hasegawa, Masatomo Touyama, Shunsuke Kondo, Hiroyuki Maeda, Mikio Suzuki, and Akira Ganaha. 2021. "Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma" Microorganisms 9, no. 3: 520. https://doi.org/10.3390/microorganisms9030520