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Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line
Fernando Gonzalez-Salinas1, Jessica Herrera-Gamboa1,2, Rocio Rojo1,3
1Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Ave. Morones Prieto 3000, Monterrey 64710, Nuevo Leon, Mexico.
Abstract:
Breast cancer is one of the leading causes of death in women around the world. Over time, many genes and mutations that are associated with the development of this disease have been identified. However, the specific role of many genes has not yet been fully elucidated. Higher ARID4B expression has been identified as a risk factor for diverse cancer types. Silencing experiments also showed that ARID4B is associated with developing cancer-associated characteristics. However, no transcriptomic studies have shown the overall cellular effect of loss of function in breast cancer in humans. This study addresses the impact of loss-of-function mutations in breast cancer MCF-7 cells. Using the CRISPR/Cas9 system, we generated mutations that caused heterozygous truncated proteins, isolating three monoclonal lines carrying insertions and deletions in ARID4B. We observed reduced proliferation and migration in in vitro experiments. In addition, from RNA-seq assays, a differential expression analysis shows known and novel deregulated cancer-associate pathways in mutated cells supporting the impact of ARID4B. For example, we found the AKT-PI3K pathway to be altered at the transcript level but through different genes than those reported for ARID4B. Our transcriptomic results also suggest new insights into the role of ARID4B in aggressiveness by the epithelial-to-mesenchymal transition and TGF-β pathways and in metabolism through cholesterol and mevalonate pathways. We also performed exome sequencing to show that no off-target effects were apparent. In conclusion, the ARID4B gene is associated with some aggressive phenotypes in breast cancer cells.
Insights
Loss of the ARID4B gene in breast cancer cells reduced proliferation and migration. This study reveals ARID4B
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Breast cancer is a leading cause of death in women globally.
- While ARID4B gene expression is linked to various cancers, its specific role in breast cancer remains unclear.
- Previous studies suggest ARID4B is associated with cancer development, but its functional impact in human breast cancer cells is not well understood.
Purpose of the Study:
- To investigate the cellular and transcriptomic effects of ARID4B loss-of-function mutations in human breast cancer MCF-7 cells.
- To identify pathways and cellular processes affected by ARID4B deficiency.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to create ARID4B loss-of-function mutations in MCF-7 cells.
- Performed in vitro proliferation and migration assays.
- Conducted RNA-sequencing (RNA-seq) for differential gene expression analysis.
- Performed exome sequencing to assess for off-target mutations.
Main Results:
- ARID4B mutations led to reduced cell proliferation and migration in vitro.
- RNA-seq analysis identified altered cancer-associated pathways, including the AKT-PI3K pathway.
- New insights into ARID4B's role in epithelial-to-mesenchymal transition, TGF-β signaling, and cholesterol/mevalonate metabolism were revealed.
- Exome sequencing confirmed no significant off-target effects from CRISPR/Cas9 editing.
Conclusions:
- The ARID4B gene plays a role in aggressive phenotypes of breast cancer cells.
- Loss of ARID4B function impacts key cellular processes relevant to cancer progression.
- Transcriptomic analysis provides a comprehensive view of ARID4B's cellular functions in breast cancer.
