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The study of effect of inhibiting the ABCB1 (P-gp) gene in reducing the resistance of cancer cells to the chemotherapeutic agent doxorubicin using CRISPR/Cas9 technology in ovarian cancer cell line A2780/ADR

Norouzi-Barough, Leyla (2018) The study of effect of inhibiting the ABCB1 (P-gp) gene in reducing the resistance of cancer cells to the chemotherapeutic agent doxorubicin using CRISPR/Cas9 technology in ovarian cancer cell line A2780/ADR. Masters thesis, qazvin university of medical sciences, Qazvin,Iran.

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Abstract

ABSTRACT 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 knockout ABCB1 (P-gp) gene expression in Adriamycin resistant (A2780/ADR) ovarian cancer cell line and evaluate the sensitivity changes to doxorubicin (adriamycin). 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. CRISPR/Cas9 efficiency to create indels in target sites was investigated by Surveyor nuclease assay. Drug sensitivity to doxorubicin was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results showed that CRISPR/Cas9 system could significantly reduce the expression of ABCB1. The dramatic decline in ABCB1 gene expression was associated with increased sensitivity of cells transfected with sgRNAs targeting ABCB1 to doxorubicin compared to the control cells. Based on the results of this study, it is concluded that the CRISPR-based systems, 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. Key Words: Drug Resistance, ABCB1, Ovarian Cancer, CRISPR/Cas9

Item Type: Thesis (Masters)
Subjects: R Medicine > RH Basic Medical Sciences > RH1001 Biochemistry & Genetics
R Medicine > RM Therapeutics. Pharmacology
Divisions: University Thesis > Faculty of Medicine
Depositing User: Medicine School Students
Date Deposited: 17 Feb 2018 08:07
Last Modified: 17 Feb 2018 08:07
URI: http://eprints.qums.ac.ir/id/eprint/7252

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