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Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells

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Abstract

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.

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Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by the Ministry of Higher Education, Malaysia under the Fundamental Research Grant Scheme FRGS/1/2016/SKK08/UM/02/16 (FP015-2016) and MyBrain15 Scholarship.

Funding

This work was supported by the Ministry of Higher Education, Malaysia under the Fundamental Research Grant Scheme FRGS/1/2016/SKK08/UM/02/16 (FP015-2016).

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Authors and Affiliations

  1. Department of Molecular Medicine, Faculty of Medicine, Universiti Malaya, 50603, Kuala Lumpur, Malaysia

    Sivasangkary Gandhi, Nurshamimi Nor Rashid, Muhammad Fazril Mohamad Razif & Shatrah Othman

  2. Drug Design and Development Research Group, Universiti Malaya, 50603, Kuala Lumpur, Malaysia

    Nurshamimi Nor Rashid & Shatrah Othman

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  1. Sivasangkary Gandhi
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  2. Nurshamimi Nor Rashid
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  3. Muhammad Fazril Mohamad Razif
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  4. Shatrah Othman
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Contributions

All authors contributed to the study conception and design. Material preparation, data collections and analysis were performed by SG. The first draft of the manuscript was written by SG while NNR, MFMR and SO contributed to the manuscript revisions. All authors read and approved the final manuscript.

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Correspondence to Nurshamimi Nor Rashid.

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Gandhi, S., Nor Rashid, N., Mohamad Razif, M.F. et al. Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells. Mol Biol Rep 48, 5121–5133 (2021). https://doi.org/10.1007/s11033-021-06509-4

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