RNF141 Knockout Huh-7 Cell Line

The RNF141 Knockout Huh-7 Cell Line is a genetically engineered human hepatocellular carcinoma cell model in which the gene encoding RING finger protein 141 (RNF141) has been disrupted using CRISPR/Cas9-mediated gene targeting. This knockout cell line provides a stable loss-of-function system for investigating the role of RNF141, a predicted E3 ubiquitin-protein ligase, in liver cancer biology and ubiquitin-dependent regulatory networks. The cell line is derived from the Huh-7 parental line and lacks functional RNF141 expression, enabling precise studies of substrate ubiquitination, protein degradation, and downstream cellular processes dependent on this ligase.

The Huh-7 cell line was originally established from a hepatocellular carcinoma of a Japanese male and is extensively employed in hepatology research, including studies of liver carcinogenesis, drug metabolism, and hepatitis virus infection. Huh-7 cells retain many hepatocyte-specific features and are highly permissive for viral replication, making them a widely accepted model for mechanistic and translational liver research. Their robust growth and well-characterized genomic landscape provide a reliable platform for knockout studies, allowing direct interrogation of gene function within a liver cancer context.

RNF141 belongs to the RING finger family of E3 ubiquitin ligases, which mediate the final transfer of ubiquitin to substrate proteins, targeting them for 26S proteasome-dependent degradation. The protein interacts with E2 ubiquitin-conjugating enzymes and proteasomal subunits to execute ubiquitin-mediated proteolysis. Although substrates remain unidentified, RNF141 is implicated in apoptosis and cell cycle regulation, consistent with its link to spermatogenesis and hepatocellular carcinoma. The ubiquitination cascade proceeds through E1 activating enzyme, E2 conjugating enzyme, and RNF141 E3 ligase, leading to polyubiquitination and proteasomal recognition. Disruption of RNF141 likely stabilizes key substrates, perturbing pathways that control cell proliferation and programmed cell death.

In Huh-7 hepatocellular carcinoma cells, RNF141 knockout offers a valuable tool to dissect how loss of this E3 ligase impacts liver cancer cell behavior. Since ubiquitin-mediated proteolysis is frequently dysregulated in cancer, altering the turnover of oncoproteins and tumor suppressors, the absence of RNF141 may reveal substrates critical for hepatocellular carcinoma growth or survival. This knockout model can be used to assess changes in cell cycle distribution, apoptotic sensitivity, and proliferation rates, providing insights into the role of RNF141 in hepatocarcinogenesis and its potential as a therapeutic target.

Researchers may employ this cell line for ubiquitination assays, co-immunoprecipitation of ubiquitin-conjugated proteins, and proteasome inhibition studies to identify RNF141 substrates. Functional readouts including CCK-8 proliferation, flow cytometry for cell cycle and apoptosis, and Western blotting can elucidate downstream effects. The model is suitable for high-throughput screens of ubiquitin-proteasome modulators in liver cancer. For detailed information, please contact Ascent Research.