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

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Mohammad Pirouzfar
Farshid Amiri
Mehdi Dianatpour
Mohammad Ali Takhshid

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.

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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
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Original articles