CRISPR/Cas۹, a new approach to successful knockdown of ABCB۱/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

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/Cas۹ genome editing technology to knockdown ABCB۱ gene expression in adriamycin resistant (A۲۷۸۰/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 ABCB۱ gene were designed in this study. Expression level of ABCB۱ was detected using quantitative real time PCR (qRT-PCR) after co-transfection of all three sgRNAs into A۲۷۸۰/ADR cell line and subsequent antibiotic selection. Drug sensitivity to doxorubicin was determined by the ۳-(۴,۵-dimethylthiazol-۲-yl)-۲,۵-diphenyltetrazolium bromide (MTT) assay. Results: The results showed that CRISPR/Cas۹ system could significantly reduce the expression of P-gp. The dramatic decline in ABCB۱ 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 A۲۷۸۰/ADR cell line resulting in restoration of nonmalignant phenotype.

Keywords:

CRISPR , Doxorubicin , Drug resistance , Ovarian cancer , P-glycoprotein