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RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53

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Abstract

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 30571950; 30170976; 30672227) and the “973” Program of China (No. 2002CB513100).

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Authors and Affiliations

  1. Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave., Wuhan, Hubei, 430030, P.R. China

    Ni Sima, Wei Wang, Dongrui Deng, Qian Xu, Jianfeng Zhou, Gang Xu, Li Meng, Yunping Lu, Shixuan Wang & Ding Ma

  2. Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, P.R. China

    Ni Sima

  3. Department of Urology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, P.R. China

    Debo Kong

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  1. Ni Sima
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  2. Wei Wang
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  3. Debo Kong
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  4. Dongrui Deng
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  5. Qian Xu
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  9. Yunping Lu
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  10. Shixuan Wang
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  11. Ding Ma
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Correspondence to Ding Ma.

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Ni Sima and Wei Wang contributed equally to this manuscript.

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Sima, N., Wang, W., Kong, D. et al. RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53. Apoptosis 13, 273–281 (2008). https://doi.org/10.1007/s10495-007-0163-8

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  • DOI: https://doi.org/10.1007/s10495-007-0163-8

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