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Updated: Jun 7, 2025
An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Cell-based assay to detect small molecules restoring levels of let-7 miRNAs
Sirinapa Szewczyk1, Brian Buckley2, Mikhail Chernov2
1Department of Molecular and Cellular Biology, Roswell Park Comprehensive Cancer Center Buffalo, NY 14263, USA.
Abstract:
Blockage of let-7 miRNA biogenesis by LIN28, or other mechanisms, results in derepression of let-7 target genes, some of which are oncogenic (e.g., MYCN) potentially contributing to tumor progression and drug resistance. We have developed a cell-based assay to identify small molecules that increase levels of mature functional let-7 miRNAs by inhibiting the function of Lin28B protein or by other means. This system consists of a reporter gene (GFP) regulated by the tTR-KRAB repressor protein which in turn is regulated by processed let-7 miRNAs. Using this system, we screened approximately 4000 small molecules and identified more than a dozen compounds capable of augmenting levels of mature let-7 miRNAs. Among those compounds, Kenpaullone and BIO were shown to increase let-7 miRNA levels with consequent suppression of MYCN protein in neuroblastoma cell lines. This novel strategy provides an additional cell-based assay for candidate cancer drug screening in a high throughput setting and will facilitate the identification of anti-cancer drugs. Moreover, this assay could be used to screen shRNA and CRISPR libraries to identify novel components of the LIN28-let-7 axis which may provide new therapeutic targets.
Insights
Researchers developed a novel cell-based assay to discover drugs that boost let-7 miRNA levels. This assay identified compounds that suppress oncogenic MYCN in neuroblastoma, offering a new avenue for cancer drug screening.
Area of Science:
- Molecular Biology
- Oncology
- Drug Discovery
Background:
- LIN28-mediated inhibition of let-7 miRNA biogenesis can lead to the upregulation of oncogenic targets like MYCN, promoting tumor progression and drug resistance.
- Identifying therapeutic strategies to restore let-7 miRNA function is crucial for cancer treatment.
Purpose of the Study:
- To develop and validate a cell-based assay for screening small molecules that enhance mature let-7 miRNA levels.
- To identify novel compounds that can inhibit LIN28B function or restore let-7 miRNA biogenesis.
- To evaluate the therapeutic potential of identified compounds in cancer models.
Main Methods:
- Development of a reporter system where GFP expression is regulated by processed let-7 miRNAs via a tTR-KRAB repressor.
- High-throughput screening of approximately 4000 small molecules using the developed cell-based assay.
- Validation of identified compounds, including Kenpaullone and BIO, for their ability to increase let-7 miRNA levels and suppress MYCN protein in neuroblastoma cell lines.
Main Results:
- Identification of over a dozen small molecules capable of augmenting mature let-7 miRNA levels.
- Kenpaullone and BIO demonstrated efficacy in increasing let-7 miRNA levels.
- Consequent suppression of MYCN protein was observed in neuroblastoma cells treated with Kenpaullone and BIO.
Conclusions:
- The developed cell-based assay is a valuable tool for high-throughput screening of anti-cancer drug candidates targeting the LIN28-let-7 axis.
- The identified compounds, Kenpaullone and BIO, show potential as therapeutic agents for cancers driven by let-7 deregulation.
- This assay can also be utilized for screening shRNA and CRISPR libraries to discover novel therapeutic targets within the LIN28-let-7 pathway.
