The EBAG9 Knockout Huh-7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for loss-of-function studies of the Estrogen Receptor-Binding Fragment-Associated Gene 9 (EBAG9) in a human hepatocellular carcinoma model. This product comprises a heterogeneous pool of Huh-7 cells with targeted disruptions in the EBAG9 gene, enabling robust analysis of gene function while minimizing clonal artifacts. The polyclonal knockout format provides a powerful tool to investigate EBAG9-mediated immune evasion mechanisms in cancer.
The Huh-7 cell line, derived from a Japanese male with well-differentiated hepatocellular carcinoma, is a widely used epithelial model for liver cancer research. These cells retain hepatocyte-like features, making them suitable for studying hepatic function, drug metabolism, and hepatotropic virus infections. The EBAG9 knockout in this background allows researchers to examine the gene’s role specifically in hepatocellular carcinoma, a tumor type where EBAG9 overexpression correlates with aggressive disease and poor clinical outcomes.
EBAG9 encodes the protein RCAS1, a transmembrane ligand that binds to the 4-1BB receptor (CD137) on activated T cells, initiating apoptosis via activation of caspase-3 and caspase-9, and thereby facilitating tumor immune escape. EBAG9 expression is transcriptionally regulated by estrogen receptor alpha (ER??), as well as by the transcription factors SP1 and AP-1, linking its activity to hormonal signaling. RCAS1 also interacts with MHC class I molecules and modulates the caspase cascade and PARP cleavage, integrating estrogen signaling with immune checkpoint regulation and T cell suppression. Additionally, downstream pro-apoptotic Bcl-2 family members are implicated in RCAS1-induced apoptosis.
In hepatocellular carcinoma, RCAS1 contributes to an immunosuppressive tumor microenvironment by inducing apoptosis in infiltrating T cells, thereby promoting tumor progression. Disrupting EBAG9 in Huh-7 cells offers a relevant model to study how loss of RCAS1 affects immune cell-mediated killing in co-culture systems, such as with Jurkat T cells, and to assess changes in tumor cell proliferation and migration. This knockout system is particularly valuable for elucidating the interplay between estrogen-driven signaling and immune evasion in liver cancer, supporting both mechanistic studies and therapeutic target validation.
Researchers can employ this polyclonal knockout product in a variety of assays, including Western blotting to confirm RCAS1 depletion, RT-qPCR for EBAG9 transcript levels, and co-culture with Jurkat T cells followed by flow cytometry-based apoptosis detection or caspase activity measurements. The cells are also suitable for cell proliferation and migration assays, RNA-seq transcriptomic profiling, and flow cytometry for T cell activation markers. These applications facilitate research into cancer immunotherapy, immune checkpoint regulation, and estrogen-responsive tumor biology. For further details, please contact Ascent Research.
