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HPV16 E6/E7-mediated regulation of PiwiL1 expression induces tumorigenesis in cervical cancer cells

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Abstract

Purpose

PiwiL1 has been reported to be over-expressed in many cancers. However, the molecular mechanism by which these proteins contribute to tumorigenesis and their regulation in cancer cells is still unclear. We intend to understand the role of PiwiL1 in tumorigenesis and also its regulation in cervical cells.

Methods

We studied the effect of loss of PiwiL1 function on tumor properties of cervical cancer cells in vitro and in vivo. Also we have looked into the effect of PiwiL1 overexpression in the malignant transformation of normal cells both in vitro and in vivo. Further RNA-seq and RIP-seq analyses were done to get insight of the direct and indirect targets of PiwiL1 in the cervical cancer cells.

Results

Here, we report that PiwiL1 is not only over-expressed, but also play a major role in tumor induction and progression. Abolition of PiwiL1 in CaSki cells led to a decrease in the tumor-associated properties, whereas, its upregulation conferred malignant transformation of normal HaCaT cells. Our study delineates a new link between HPV oncogenes, E6 and E7 with PiwiL1. p53 and E2F1 directly bind and differentially regulate PiwiL1 promoter in a context-dependant manner. Further, RNA-seq together with RIP-RNA-seq suggested a strong and direct role for PiwiL1 in promoting metastasis in cervical cancer cells.

Conclusion

Our study demonstrates that PiwiL1 act as an oncogene in cervical cancer by inducing tumor-associated properties and EMT pathway. The finding that HPV oncogenes, E6/E7 can positively regulate PiwiL1 suggests a possible mechanism behind HPV-mediated tumorigenesis in cervical cancer.

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Data availability

All data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We acknowledge the University Grants Commission (UGC), Government of India, for the Senior Research Fellowship to MK and PSR. We want to thank all Neuro Stem Cell Biology Lab (RGCB) members and Jayasree R for their various technical supports. We thank Dr. Zhi-Ming Zheng for providing the pZMZ70 plasmid. We acknowledge Dr. Arumugam Rajavelu and Dr. Tessy Thomas Maliekkal for their valuable suggestions in experiment design. We thank Ms. Jayalekshmi V S and Ms. Neethu Krishnan for their help in histological work. We are extremely thankful to Dr. Sumitra Shankar for the critical reading of the manuscript. We acknowledge Clevergene Bicorp Pvt. Ltd, Bangalore for total and RIP RNA sequencing.

Funding

This work was supported by Institutional intramural funding. M.K [961/[CSIR-UGC NET DEC 2016] and P.S.R [888/[CSIR-UGC NET DEC 2018]. have received research support from UGC, Govt. of India.

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Authors and Affiliations

  1. Cancer Research Program-12, Rajiv Gandhi Centre for Biotechnology (DBT-RGCB), Thycaud. P.O., Thiruvananthapuram-14, Kerala, India

    Midhunaraj Kunnummal, Pooja Sherly Raveendran, Sheri Vidya Rani, Mary Angelin, Joby Issac & Ani V. Das

  2. Manipal Academy of Higher Education, Tiger Circle Road, Madhav Nagar, Manipal, Karnataka, 576104, India

    Midhunaraj Kunnummal & Pooja Sherly Raveendran

  3. Neuro Stem Cell Biology Laboratory, Neurobiology Division, Rajiv Gandhi Centre for Biotechnology (DBT-RGCB), Thiruvananthapuram, Kerala, 695 014, India

    Budhaditya Basu, Riya Ann Paul & Jackson James

  4. Regional Centre for Biotechnology (DBT-RCB), Faridabad, Haryana, 121001, India

    Budhaditya Basu

  5. Department of Biotechnology, University of Kerala, Thiruvananthapuram, 695011, Kerala, India

    Riya Ann Paul

  6. Bioscience Research and Training Centre, Kerala Veterinary and Animal Science University, Thonnakkal, Thiruvananthapuram, 695317, Kerala, India

    Krithiga Kuppusamy

Authors
  1. Midhunaraj Kunnummal
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  2. Pooja Sherly Raveendran
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  10. Ani V. Das
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Contributions

All authors contributed to the study conception and design. The first draft of the manuscript was written by MK and all authors commented on previous versions of the manuscript; MK: Data curation, writing–original draft, Data visualization; PSR: assisted with the in vitro experiments; BB: Bioinformatics, Data visualization and helped in in vivo experiments; SVR: performed qRT-PCR analysis; RAP: helped in in vivo experiments; KK: histopathology data analysis and interpretation; MA: assisted with western blot; JI: assisted with immunocytochemistry; JJ: Intellectually contributed to experimental design AVD: Conceptualization, Designing experiments, Interpreted data, Critical revision. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ani V. Das.

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Ethics approval

All in vivo experiments followed the institutional animal ethical committee (IAEC/815/ANI/2020) guidelines.

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The authors have no relevant financial or non-financial interests to disclose.

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Supplementary Information

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Supplementary file1 (PDF 2043 KB)

Supplementary file2 (PDF 285 KB)

Supplementary file3 (PDF 198 KB)

Supplementary file4 (PDF 10 KB)

Supplementary file5 (PDF 13 KB)

13402_2023_904_MOESM6_ESM.csv

Supplementary file6 (CSV 543 KB) Supplementary Table 5: Significant upregulated genes in PIWIL1 overexpressed HaCaT cells

13402_2023_904_MOESM7_ESM.csv

Supplementary file7 (CSV 515 KB) Supplementary Table 6: Significant downregulated genes in PIWIL1 overexpressed HaCaT cells

Supplementary file8 (CSV 55 KB) Supplementary Table 7: Gene Ontology enrichment analysis of PIWIL1 upregulated genes

Supplementary file9 (CSV 25 KB) Supplementary Table 8: Gene Ontology enrichment analysis of PIWIL1 downregulated genes

13402_2023_904_MOESM10_ESM.csv

Supplementary file10 (CSV 80 KB) Supplementary Table 9: Gene Set Enrichment Analysis between HaCaT control and HaCaT-PIWIL1 overexpressed cells

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Supplementary file11 (CSV 1 KB) Supplementary Table 10: The list of genes common in PIWIL1 RIP- Seq data and PIWIL1 positively regulated transcripts

13402_2023_904_MOESM12_ESM.csv

Supplementary file12 (CSV 4 KB) Supplementary Table 11: The list of genes common in PIWIL1 RIP- Seq data and PIWIL1 negatively regulated transcripts

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Kunnummal, M., Raveendran, P.S., Basu, B. et al. HPV16 E6/E7-mediated regulation of PiwiL1 expression induces tumorigenesis in cervical cancer cells. Cell Oncol. (2023). https://doi.org/10.1007/s13402-023-00904-8

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