Crispr/Cas9, a New Approach to Successful Knockdown of Abcb1/P-Glycoprotein and Reversal of Chemosensitivity in Human Epithelial Ovarian Cancer Cell Line Publisher



Norouzibarough L¹ ; Sarookhani M² ; Salehi R³ ; Sharifi M³ ; Moghbelinejad S⁴ Show Affiliations
Source: Iranian Journal of Basic Medical Sciences Published:2018

Abstract

Objective(s): Multidrug resistance (MDR) is a major obstacle in the successful chemotherapy of ovarian cancer. Inhibition of P-glycoprotein (P-gp), a member of ATP-binding cassette (ABC) transporters, is a well-known strategy to overcome MDR in cancer. The aim of this study was to investigate the efficiency and ability of CRISPR/Cas9 genome editing technology to knockdown ABCB1 gene expression in adriamycin resistant (A2780/ADR) ovarian cancer cell line and evaluate the sensitivity changes to doxorubicin. Materials and Methods: Three single-guide RNAs (sgRNAs) targeting the fourth and fifth exons of human ABCB1 gene were designed in this study. Expression level of ABCB1 was detected using quantitative real time PCR (qRT-PCR) after co-transfection of all three sgRNAs into A2780/ADR cell line and subsequent antibiotic selection. Drug sensitivity to doxorubicin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The results showed that CRISPR/Cas9 system could significantly reduce the expression of Pgp. The dramatic decline in ABCB1 gene expression was associated with increased sensitivity of cells transfected with sgRNAs to doxorubicin. Conclusion: Based on the results of this study, it is concluded that the CRISPR-based systems, used in the present study, effectively down-regulated the target gene and acted as an ideal and cost-effective tool for gene editing of A2780/ADR cell line resulting in restoration of nonmalignant phenotype. © 2018, Mashhad University of Medical Sciences. All rights reserved.