Identification of chemical inhibitors targeting long noncoding RNA through gene signature-based high throughput screening

Scalable methods for functionally high-throughput screening of RNA-targeting small molecules are currently limited. Here, an RNA knockdown gene signature and high-throughput sequencing-based high-throughput screening (HTS²) were integrated to identify RNA-targeting compounds. We first generated a gene signature characterizing the knockdown of the long non-coding RNA LINC00973. Then, screening of 8199 compounds by HTS² assay identified that treatments of Hesperadin and GSK1070916 significantly mimic the expression pattern of the LINC00973 knockdown gene signature. Functionally, cell phenotype changes after treatments of these two compounds also mimic the losing function of LINC00973 in multiple types of cancer cells. Mechanistically, the inhibitory action of these two compounds on LINC00973 primarily operates via the AURKB-mediated MAPK signaling pathway, resulting in reduced expression of the transcription factor c-Jun. Consequently, this leads to the suppression of LINC00973 transcription. Moreover, these two compounds significantly inhibit xenograft tumor growth in vivo. Clinically, we further found that breast tumors with high expression of LINC00973 also show relatively high expression of AURKB or JUN, and vice versa. In summary, we established a novel high-throughput screening strategy to identify small molecules capable of targeting RNA, provided two promising compounds targeting LINC00973 and further shed light on the underlying transcriptional upregulation mechanism of LINC00973 within cancer cells.