CRISPR/Cas9-Mediated Knockout of ZFP36L1 Impairs Cell Proliferation, Alters Cell-Cycle Progression, and Enhances DNA Damage Responses in MDA-MB-231 Triple-Negative Breast Cancer Cells
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Background
Zinc finger protein 36-like 1 (ZFP36L1) is an AU-rich element-binding RNA-binding protein that regulates post-transcriptional gene expression and has been implicated in tumor progression, cell-cycle regulation, and DNA damage responses. However, its functional role in triple-negative breast cancer (TNBC) remains poorly understood. This study investigated the effects of CRISPR/Cas9-mediated ZFP36L1 knockout on cell proliferation, doxorubicin (DOX) sensitivity, cell-cycle progression, and DNA damage responses in MDA-MB-231 TNBC cells.
Methods
Wild-type (WT) and CRISPR/Cas9-generated ZFP36L1 knockout (KO) MDA-MB-231 cells were cultured under standard conditions. Cellular proliferation was evaluated by cell counting over three weeks. Cell viability following DOX treatment was determined using the MTT assay, and half-maximal inhibitory concentration (IC 50 ) values were calculated. Cell-cycle distribution was assessed by propidium iodide flow cytometry after 24 h of DOX exposure, while DNA damage was quantified by γ-H2AX flow cytometric analysis. Statistical significance was determined using Student’s t -test with P < 0.05 considered significant.
Results
ZFP36L1 knockout reduced the proliferative capacity of MDA-MB-231 cells compared with WT cells. Both cell lines exhibited dose-dependent decreases in viability following DOX treatment. KO cells demonstrated a higher mean IC 50 than WT cells (9.64 vs. 8.40 μM), indicating a trend toward reduced DOX sensitivity; however, this difference was not statistically significant ( P = 0.569). Flow cytometric analysis revealed enhanced accumulation of KO cells in the S and G 2 /M phases following DOX treatment, suggesting altered cell-cycle checkpoint regulation. Furthermore, KO cells exhibited elevated basal γ-H2AX expression and greater DOX-induced γ-H2AX accumulation than WT cells, indicating increased DNA damage and impaired maintenance of genomic stability.
Conclusions
CRISPR/Cas9-mediated loss of ZFP36L1 suppresses proliferation, alters cell-cycle checkpoint dynamics, and enhances DNA damage accumulation in MDA-MB-231 TNBC cells. These findings indicate that ZFP36L1 plays a context-dependent role in regulating genomic stability and cellular responses to genotoxic stress, highlighting its potential as a biomarker and therapeutic target in triple-negative breast cancer.
