Comparative effectiveness of 7 major human let-7-5p isoforms to modulate target gene expression in liver cells.

Dysregulation of efflux ATP-binding cassette (ABC) transporters often confers multidrug resistance, presenting significant challenges in treating various diseases (eg, hepatocellular carcinoma [HCC]). The let-7-5p microRNAs (miRNAs), commonly downregulated in HCC, have established roles in controlling post-transcriptional gene regulation of ABC transporters (eg, multidrug resistance-associated protein 5 MRP5/ABCC5) and some oncogenes (eg, RNA-binding protein LIN28B). Although previous research has demonstrated the potential of particular let-7-5p isoforms to regulate ABC transporters and inhibit HCC cell viability, the comparative efficacy of let-7-5p isoforms whose sequences differ in several nucleosides is unknown. This study was to compare the effectiveness of 7 major let-7-5p isoforms (let-7a to let-7g) to regulate ABCC5 and LIN28B targets and inhibit HCC cell viability in vitro by using novel bioengineered RNA let-7-5p (BioRNA/let-7-5p) agents. Release of let-7-5p isoforms from individual BioRNA/let-7-5p molecules in Huh7, HepG2, and Hep3B cells was validated. Efficacy of BioRNA/let-7-5p isoforms to repress ABCC5/MRP5 and LIN28B protein levels was found to be target dependent; among them, let-7c and let-7d-5p exhibited broader regulatory efficacy against ABCC5/MRP5, while let-7d-5p emerged as the most potent suppressor of LIN28B, generally in accordance with let-7-5p abundance and target complementarity. By contrast, let-7-5p isoforms showed minimal impact on ABCC2/MRP2 and ABCC4/MRP4 protein levels. In addition, let-7-5p isoforms showed variable efficacy to inhibit the viability of different HCC cells. Together, our studies established the functional differences of let-7-5p isoforms in regulating target gene expression and inhibiting HCC cell viability, providing insights into intrinsic differences of miRNA isoforms to inform rational development of miRNA therapeutics or combination therapy. SIGNIFICANCE STATEMENT: Using novel bioengineered RNA agents, this study established the functional differences of 7 major human let-7-5p isoforms to control target gene expression and hepatocellular carcinoma cell viability in vitro. These findings demonstrate the potential of bioengineered RNA molecules to interrogate post-transcriptional gene regulation mechanisms, highlighting specific let-7-5p isoforms to modulate transporter and oncogene expression toward the development of improved therapies.