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Updated: Jul 31, 2025
Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype
Published on: June 17, 2022
Protocol to screen for Sorafenib resistance regulators using pooled lentiviral shRNA library and a
Ruize Gao1, Fengyuan Tang1, Gerhard Christofori1
1Department of Biomedicine, University of Basel, CH4058 Basel, Switzerland.
Abstract:
Approaches to study therapy resistance in HCC (hepatocellular carcinoma) are limited, especially when using HCC models in vitro. Here, we present a protocol to establish an in vitro Sorafenib-resistant human HCC cell model and conduct an shRNA-mediated synthetic lethal screen in established Sorafenib-resistant HCC cell lines to identify critical regulators of Sorafenib resistance. We describe steps for RNA sequencing and functional analysis to reveal the mode of action of potential candidates in conferring therapy resistance to HCC cells. For complete details on the use and execution of this protocol, please refer to Gao et al. (2021a)1 and Gao et al. (2021b).2.
Insights
This study establishes a novel in vitro model for hepatocellular carcinoma (HCC) therapy resistance. Researchers identified key regulators of Sorafenib resistance in HCC cells using a synthetic lethal screen.
Area of Science:
- Oncology
- Molecular Biology
- Drug Resistance
Background:
- Therapy resistance in hepatocellular carcinoma (HCC) is a significant clinical challenge.
- Existing in vitro models for studying HCC resistance are limited.
- Sorafenib is a standard targeted therapy for advanced HCC.
Purpose of the Study:
- To develop a protocol for establishing an in vitro Sorafenib-resistant human HCC cell model.
- To identify critical regulators of Sorafenib resistance in HCC.
- To elucidate the mode of action of identified resistance regulators.
Main Methods:
- Establishment of an in vitro Sorafenib-resistant HCC cell line.
- shRNA-mediated synthetic lethal screening in resistant HCC cells.
- RNA sequencing and functional analysis of candidate genes.
Main Results:
- A reproducible protocol for generating Sorafenib-resistant HCC models was developed.
- The study identified potential key regulators involved in HCC therapy resistance.
- Functional analyses provided insights into the mechanisms of Sorafenib resistance.
Conclusions:
- The developed in vitro model facilitates the study of hepatocellular carcinoma therapy resistance.
- The synthetic lethal screen successfully identified novel targets regulating Sorafenib resistance.
- This research provides a foundation for developing strategies to overcome HCC drug resistance.
