Activities of E6 Protein of Human Papillomavirus 16 Asian Variant on miR-21 Up-regulation and Expression of Human Immune Response Genes

  • Chopjitt, Peechanika (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Pientong, Chamsai (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Bumrungthai, Sureewan (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Kongyingyoes, Bunkerd (Department of Pharmacology, Faculty of Medicine, Khon Kaen University) ;
  • Ekalaksananan, Tipaya (Department of Microbiology, Faculty of Medicine, Khon Kaen University)
  • Published : 2015.05.18


Background: Variants of human papillomavirus (HPV) show more oncogenicity than do prototypes. The HPV16 Asian variant (HPV16As) plays a major role in cervical cancer of Asian populations. Some amino acid changes in the E6 protein of HPV16 variants affect E6 functions such as p53 interaction and host immune surveillance. This study aimed to investigate activities of HPV16As E6 protein on modulation of expression of miRNA-21 as well as interferon regulatory factors (IRFs) 1, 3, 7 and c-fos. Materials and Methods: Vectors expressing E6 protein of HPV16As (E6D25E) or HPV16 prototype (E6Pro) were constructed and transfected into C33A cells. HCK1T cells expressing E6D25E or E6Pro were established by transducing retrovirus-containing E6D25E or 16E6Pro. The E6AP-binding activity of E6 and proliferation of the transfected C33A cells were determined. MiR-21 and mRNA of interesting genes were detected in the transfected C33A cells and/or the HCK1T cells, with or without treatment by culture medium from HeLa cells (HeLa-CM). Results: E6D25E showed binding activity with E6AP similar to that of E6Pro. Interestingly, E6D25E showed a higher activity of miR-21 induction than did E6Pro in C33A cells expressing E6 protein. This result was similar to the HCK1T cells expressing E6 protein, with HeLa-CM treatment. The miR-21 up-regulation significantly corresponded to its target expression. Different levels of expression of IRFs were also observed in the HCK1T cells expressing E6 protein. Interestingly, when treated with HeLa-CM, IRFs 1, 3 and 7 as well as c-fos were significantly suppressed in the HCK1T cells expressing E6D25E, whereas those in the HCK1T cells expressing E6Pro were induced. A similar situation was seen for IFN-${\alpha}$ and IFN-${\beta}$. Conclusions: E6D25E of the HPV16As variant differed from the E6 prototype in its activities on epigenetic modulation and immune surveillance and this might be a key factor for the important role of this variant in cervical cancer progression.


HPV16 Asian variant;E6D25E;miR-21, IRFs;c-fos


  1. Barnard P, McMillan NA (1999). The human papillomavirus E7 oncoprotein abrogates signaling mediated by interferon-alpha. Virology, 259, 305-13.
  2. Chang YJ, Chen HC, Pan MH, et al (2013). Intratypic variants of human papillomavirus type 16 and risk of cervical neoplasia in Taiwan. J Med Virol, 85, 1567-76.
  3. Choi BS, Kim SS, Yun H, et al (2007). Distinctive distribution of HPV16 E6 D25E and E7 N29S intratypic Asian variants in Korean commercial sex workers. J Med Virol, 79, 426-30.
  4. Chopjitt P, Ekalaksananan T, Pientong C, et al (2009). Prevalence of human papillomavirus type 16 and its variants in abnormal squamous cervical cells in Northeast Thailand. Int J Infect Dis, 13, 212-9.
  5. Cornet I, Gheit T, Franceschi S, et al (2012). Human papillomavirus type 16 genetic variants: phylogeny and classification based on E6 and LCR. J Virol, 86, 6855-61.
  6. Gheit T, Cornet I, Clifford GM, et al (2011). Risks for persistence and progression by human papillomavirus type 16 variant lineages among a population-based sample of Danish women. Cancer Epidemiol Biomarkers Prev, 20, 1315-21.
  7. Hang D, Gao L, Sun M, et al (2014). Functional effects of sequence variations in the E6 and E2 genes of human papillomavirus 16 European and Asian variants. J Med Virol, 86, 618-26.
  8. Howie HL, Katzenellenbogen RA, Galloway DA (2009). Papillomavirus E6 proteins. Virology, 384, 324-34.
  9. Huibregtse JM, Scheffner M, Howley PM (1993). Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins. Mol Cell Biol, 13, 4918-27.
  10. Kang S, Jeon YT, Kim JW, et al (2005). Polymorphism in the E6 gene of human papillomavirus type 16 in the cervical tissues of Korean women. Int J Gynecol Cancer, 15, 107-12.
  11. Kavitha N, Vijayarathna S, Jothy SL, et al (2014). MicroRNAs: biogenesis, roles for carcinogenesis and as potential biomarkers for cancer diagnosis and prognosis. Asian Pac J Cancer Prev, 15, 7489-97.
  12. Kiyono T, Hiraiwa A, Fujita M, et al (1997). Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proc Natl Acad Sci USA, 94, 11612-6.
  13. Li S, Liang Z, Xu L, et al (2012). MicroRNA-21: a ubiquitously expressed pro-survival factor in cancer and other diseases. Mol Cell Biochem, 360, 147-58.
  14. Li Y, Liu J, Yuan C, et al (2010). High-risk human papillomavirus reduces the expression of microRNA-218 in women with cervical intraepithelial neoplasia. J Int Med Res, 38, 1730-6.
  15. Li Y, Wang F, Xu J, et al (2011). Progressive miRNA expression profiles in cervical carcinogenesis and identification of HPV-related target genes for miR-29. J Pathol, 224, 484-95.
  16. Lui WO, Pourmand N, Patterson BK, et al (2007). Patterns of known and novel small RNAs in human cervical cancer. Cancer Res, 67, 6031-43.
  17. Martinez I, Gardiner AS, Board KF, et al (2008). Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene, 27, 2575-82.
  18. Mosmann JP, Monetti MS, Frutos MC, et al (2015). Mutation detection of E6 and LCR genes from HPV 16 associated with carcinogenesis. Asian Pac J Cancer Prev, 16, 1151-7.
  19. Narisawa-Saito M, Yoshimatsu Y, Ohno S, et al (2008). An in vitro multistep carcinogenesis model for human cervical cancer. Cancer Res, 68, 5699-705.
  20. Park JS, Kim EJ, Kwon HJ, et al (2000). Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis. J Biol Chem, 275, 6764-9.
  21. Pientong C, Wongwarissara P, Ekalaksananan T, et al (2013). Association of human papillomavirus type 16 long control region mutation and cervical cancer. Virol J, 10, 30.
  22. Rao Q, Shen Q, Zhou H, et al (2012). Aberrant microRNA expression in human cervical carcinomas. Med Oncol, 29, 1242-8.
  23. Ren C, Cheng X, Lu B, et al (2013). Activation of interleukin-6/signal transducer and activator of transcription 3 by human papillomavirus early proteins 6 induces fibroblast senescence to promote cervical tumourigenesis through autocrine and paracrine pathways in tumour microenvironment. Eur J Cancer, 49, 3889-99.
  24. Ronco LV, Karpova AY, Vidal M, et al (1998). Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev, 12, 2061-72.
  25. Sasagawa T, Takagi H, Makinoda S (2012). Immune responses against human papillomavirus (HPV) infection and evasion of host defense in cervical cancer. J Infect Chemother, 18, 807-15.
  26. Schiffman M, Rodriguez AC, Chen Z, et al (2010). A population-based prospective study of carcinogenic human papillomavirus variant lineages, viral persistence, and cervical neoplasia. Cancer Res, 70, 3159-69.
  27. Sheedy FJ, Palsson-McDermott E, Hennessy EJ, et al (2010). Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21. Nat Immunol, 11, 141-7.
  28. Shukla S, Shishodia G, Mahata S, et al (2010). Aberrant expression and constitutive activation of STAT3 in cervical carcinogenesis: implications in high-risk human papillomavirus infection. Mol Cancer, 9, 282.
  29. Smith EJ, Marie I, Prakash A, et al (2001). IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein. J Biol Chem, 276, 8951-7.
  30. Sobti RC, Singh N, Hussain S, et al (2009). Overexpression of STAT3 in HPV-mediated cervical cancer in a north Indian population. Mol Cell Biochem, 330, 193-9.
  31. Suthipintawong C, Siriaunkgul S, Tungsinmunkong K, et al (2011). Human papilloma virus prevalence, genotype distribution, and pattern of infection in Thai women. Asian Pac J Cancer Prev, 12, 853-6.
  32. Takaoka A, Tamura T, Taniguchi T (2008). Interferon regulatory factor family of transcription factors and regulation of oncogenesis. Cancer Sci, 99, 467-78.
  33. Taylor WR, Stark GR (2001). Regulation of the G2/M transition by p53. Oncogene, 20, 1803-15.
  34. Tungteakkhun SS, Duerksen-Hughes PJ (2008). Cellular binding partners of the human papillomavirus E6 protein. Arch Virol, 153, 397-408.
  35. Um SJ, Rhyu JW, Kim EJ, et al (2002). Abrogation of IRF-1 response by high-risk HPV E7 protein in vivo. Cancer Lett, 179, 205-12.
  36. Walboomers JM, Jacobs MV, Manos MM, et al (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol, 189, 12-9.<12::AID-PATH431>3.0.CO;2-F
  37. Wang X, Tang S, Le SY, et al (2008). Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One, 3, 2557.
  38. Xi LF, Koutsky LA, Hildesheim A, et al (2007). Risk for high-grade cervical intraepithelial neoplasia associated with variants of human papillomavirus types 16 and 18. Cancer Epidemiol Biomarkers Prev, 16, 4-10.
  39. Xie H, Zhao Y, Caramuta S, et al (2012). miR-205 expression promotes cell proliferation and migration of human cervical cancer cells. PLoS One, 7, 46990.
  40. Xu YZ, Xi QH, Ge WL, et al (2013). Identification of serum microRNA-21 as a biomarker for early detection and prognosis in human epithelial ovarian cancer. Asian Pac J Cancer Prev, 14, 1057-60.
  41. Yablonska S, Hoskins EE, Wells SI, et al (2013). Identification of miRNAs dysregulated in human foreskin keratinocytes (HFKs) expressing the human papillomavirus (HPV) Type 16 E6 and E7 oncoproteins. Microrna, 2, 2-13.
  42. Yamada T, Manos MM, Peto J, et al (1997). Human papillomavirus type 16 sequence variation in cervical cancers: a worldwide perspective. J Virol, 71, 2463-72.
  43. Yao Q, Cao S, Li C, et al (2011). Micro-RNA-21 regulates TGF-beta-induced myofibroblast differentiation by targeting PDCD4 in tumor-stroma interaction. Int J Cancer, 128, 1783-92.
  44. Yao Q, Xu H, Zhang QQ, et al (2009). MicroRNA-21 promotes cell proliferation and down-regulates the expression of programmed cell death 4 (PDCD4) in HeLa cervical carcinoma cells. Biochem Biophys Res Commun, 388, 539-42.
  45. Yi JW, Jang M, Kim SJ, et al (2013). Degradation of p53 by natural variants of the E6 protein of human papillomavirus type 16. Oncol Rep, 29, 1617-22.
  46. Young MR, Santhanam AN, Yoshikawa N, et al (2010). Have tumor suppressor PDCD4 and its counteragent oncogenic miR-21 gone rogue? Mol Interv, 10, 76-9.
  47. Zehbe I, Wilander E, Delius H, et al (1998). Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype. Cancer Res, 58, 829-33.
  48. Zhang EY, Tang XD (2012). Human papillomavirus type 16/18 oncoproteins: potential therapeutic targets in non-smoking associated lung cancer. Asian Pac J Cancer Prev, 13, 5363-9.
  49. Zhang L, Pagano JS (1997). IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency. Mol Cell Biol, 17, 5748-57.

Cited by

  1. MicroRNA-21 regulates the proliferation and apoptosis of cervical cancer cells via tumor necrosis factor-α vol.16, pp.4, 2017,
  2. Effects of arecoline on proliferation of oral squamous cell carcinoma cells by dysregulating c-Myc and miR-22, directly targeting oncostatin M vol.13, pp.1, 2018,