Cell-based assay to detect small molecules restoring levels of let-7 miRNAs.

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.