CRISPR/Cas9-mediated knockout of TopBP1 shifts the Bax/Bcl-2 balance toward apoptosis in MCF7 breast cancer cells.
Source: PubMed, NCBI / U.S. National Library of Medicine
Breast cancer (BC) is the most prevalent cancer among women and the second leading cause of cancer-related deaths globally, after lung cancer. Despite advances in treatment, BC remains a major contributor to cancer mortality worldwide, underscoring the need for innovative therapeutic approaches. The TopBP1 (DNA Topoisomerase II Binding Protein 1) gene, involved in DNA damage response and cell cycle regulation, has been associated with cancer progression and resistance to chemotherapy. This study investigates the potential of using CRISPR/Cas9 technology to knockout the TopBP1 gene as a novel strategy in breast cancer research. A pair of guide RNAs (gRNAs) was specifically designed to target the TopBP1 gene, inducing the deletion of exon 4. These gRNAs were transfected into the MCF7 breast cancer cell line, and the efficacy of genomic editing was validated using PCR and Sanger sequencing. Subsequent analyses employing real-time PCR and Western blotting were conducted to investigate the downstream effects of this genetic modification on gene expression. The CRISPR/Cas9 system successfully knocked out exon 4 of the TopBP1 gene in MCF7 breast cancer cells, as validated by PCR and Sanger sequencing. Real-time PCR analysis revealed a significant increase in Bax expression and a decrease in Bcl-2 expression in the knockout cells compared to controls. These changes indicate enhanced apoptotic activity following TopBP1 knockout, suggesting that MCF7 cells may become more sensitive to
Abstract
Breast cancer (BC) is the most prevalent cancer among women and the second leading cause of cancer-related deaths globally, after lung cancer. Despite advances in treatment, BC remains a major contributor to cancer mortality worldwide, underscoring the need for innovative therapeutic approaches. The TopBP1 (DNA Topoisomerase II Binding Protein 1) gene, involved in DNA damage response and cell cycle regulation, has been associated with cancer progression and resistance to chemotherapy. This study investigates the potential of using CRISPR/Cas9 technology to knockout the TopBP1 gene as a novel strategy in breast cancer research. A pair of guide RNAs (gRNAs) was specifically designed to target the TopBP1 gene, inducing the deletion of exon 4. These gRNAs were transfected into the MCF7 breast cancer cell line, and the efficacy of genomic editing was validated using PCR and Sanger sequencing. Subsequent analyses employing real-time PCR and Western blotting were conducted to investigate the downstream effects of this genetic modification on gene expression. The CRISPR/Cas9 system successfully knocked out exon 4 of the TopBP1 gene in MCF7 breast cancer cells, as validated by PCR and Sanger sequencing. Real-time PCR analysis revealed a significant increase in Bax expression and a decrease in Bcl-2 expression in the knockout cells compared to controls. These changes indicate enhanced apoptotic activity following TopBP1 knockout, suggesting that MCF7 cells may become more sensitive to apoptosis. Overall, the findings support the hypothesis that targeting TopBP1 could play a critical role in promoting cell death in breast cancer, potentially offering a new therapeutic strategy. This study successfully employed the CRISPR/Cas9 system to knockout exon 4 of the TopBP1 gene in MCF7 breast cancer cells, resulting in reduced TopBP1 expression. The subsequent increase in the pro-apoptotic Bax gene and decrease in the anti-apoptotic Bcl-2 gene suggest that targeting TopBP1 could enhance apoptosis in breast cancer cells, offering a promising alternative to conventional treatments. Further research is necessary to fully explore the therapeutic potential of this approach.