Authors: Pirouzfar, Mohammad
Amiri, Farshid
Dianatpour, Mehdi
Takhshid, Mohammad Ali
Title: CRISPR/Cas9-mediated knockout of MLL5 enhances apoptotic effect of cisplatin in HeLa cells in vitro
Language (ISO): en
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. ‎
Subject Headings: Cervical cancer
Mixed lineage leukemia protein
E6 protein
Issue Date: 2020-01-23
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Appears in Collections:Original Articles 2020

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