CRISPR/Cas9-mediated knockout of MLL5 enhances apoptotic effect of cisplatin in HeLa cells in vitro

Authors

  • Mohammad Pirouzfar Diagnostic Laboratory Sciences and Technology Research Center, Paramedical School, ‎Shiraz University of Medical Sciences, Meshkinfam Street, Shiraz, Iran http://orcid.org/0000-0002-8061-2503
  • Farshid Amiri Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran http://orcid.org/0000-0001-6623-0026
  • Mehdi Dianatpour Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, Tel-Fax: +98 7132349610, E-mail: Dianatpour@sums.ac.ir http://orcid.org/0000-0003-1217-9477
  • Mohammad Ali Takhshid Diagnostic Laboratory Sciences and Technology Research Center, Paramedical School, ‎Shiraz University of Medical Sciences, Meshkinfam Street, Shiraz, Iran, Tel-Fax: +987132289113, E-mail: takhshidma@sums.ac.ir http://orcid.org/0000-0003-0246-3765

DOI:

https://doi.org/10.17179/excli2019-1957

Keywords:

cervical cancer, CRISPR/Cas9, Mixed lineage leukemia protein, E6 protein, Cisplatin

Abstract

Mixed lineage leukemia 5 (MLL5) transactivates the expression of E6 and E7 oncogenes in cervical cancer cells. In this study, we utilized ‎CRISPR/Cas9 system with the aim to target HPV-E6 and MLL5 to enhance apoptosis efficiency in HPV-18 positive HeLa cells and to improve chemotherapeutic efficacy of Cisplatin as the most common anticancer drug, used for cervical cancer. ‎sgRNAs against MLL5 and E6 were designed and cloned into PX458 plasmid vector. ‎Real-time ‎PCR was used to determine knockout expression of MLL5 and E6 following, ‎transfection with cloned plasmids. Cell viability and apoptosis were evaluated, using ‎Dimethyl-thiazolyl diphenyl tetrazolium bromide (MTT) ‎assay and Annexin V flow cytometry. ‎‏Cellular p‎53 level was measured, using enzyme linked immune sorbent assay (ELISA).‏ Real-time ‎PCR indicated the downregulation of E6 and MLL5 in the transfected cells. ‎A significant increase in the accumulation of P53 was observed due to targeting MLL5 and E6 genes. MTT and ‎apoptosis assays showed a significant decrease in cell viability and enhanced apoptosis rate of ‎transfected cells. Combination therapy showed that targeting E6 and MLL5 enhanced ‎apoptotic effect of Cisplatin in MLL5 knockout cells in a synergistic manner. ‎‏The results suggest that CRISPR/Cas9 targeting of E6 and MLL5 genes can increase‎ apoptotic effects of Cisplatin and can be considered as a ‎‎potential combination therapy for the treatment of HPV-‎related cervical cancer. ‎

Downloads

Published

2020-01-23

How to Cite

Pirouzfar, M., Amiri, F., Dianatpour, M., & Takhshid, M. A. (2020). CRISPR/Cas9-mediated knockout of MLL5 enhances apoptotic effect of cisplatin in HeLa cells in vitro. EXCLI Journal, 19, 170–182. https://doi.org/10.17179/excli2019-1957

Issue

Vol. 19 (2020)

Section

Original articles

License

Authors who publish in this journal agree to the following terms:

  • The authors keep the copyright and grant the journal the right of first publication under the terms of the Creative Commons Attribution license, CC BY 4.0. This licencse permits unrestricted use, distribution and reproduction in any medium, provided that the original work is properly cited.
  • The use of general descriptive names, trade names, trademarks, and so forth in this publication, even if not specifically identified, does not imply that these names are not protected by the relevant laws and regulations.
  • Because the advice and information in this journal are believed to be true and accurate at the time of publication, neither the authors, the editors, nor the publisher accept any legal responsibility for any errors or omissions presented in the publication. The publisher makes no guarantee, express or implied, with respect to the material contained herein.
  • The authors can enter into additional contracts for the non-exclusive distribution of the journal's published version by citing the initial publication in this journal (e.g. publishing in an institutional repository or in a book).