Efficacy of Biologic let-7-5p Isoforms in the Modulation of Target Gene Expression in HCC Cells

Abstract ID 90759 Poster Board 228 Advancing our understanding of epigenetic regulation in drug metabolism and pharmacokinetics (DMPK) is critical to designing the next generation of safe and effective therapeutics. Multidrug resistance (MDR) is a major barrier in the treatment of hepatocellular carcinoma (HCC) and other cancers and is characterized by the overexpression of efflux transporters such as ABCC1/MRP1, ABCC2/MRP2, and ABCC5/MRP5 that are critical to the absorption, distribution, metabolism, and excretion (ADME) processes of chemotherapeutic drugs. Endogenous microRNA (miRNA) are small noncoding RNAs that are critical posttranscriptional regulators of a wide variety of cellular processes, capable of targeting multiple mRNA transcripts to modulate drug transport and disease progression. The let-7-5p miRNA family consists of several unique isoforms (let-7a, b, c, d, e, f, g, and i) that have demonstrated potential in previous experiments inhibiting HCC viability and negatively regulating the expression of ADME genes implicated in MDR. To further elucidate the therapeutic potential and efficacy of individual let-7-5p isoforms against HCC, the current study utilized a novel in vivo fermentation based, RNA bioengineering approach to produce unique biologic let-7-5p miRNAs (BioRNA/let-7-5p) in high yield. When analyzed by Western blot, three human HCC cell lines (Hep3B, Huh7, and HepG2) showed high basal protein expression of several potential let-7-5p targets (ABCC1-5, Lin28B) and were selected as suitable in vitro models for future study. HCC cell lines were transfected with individual BioRNA/let-7-5p agents for 48-h or 72-h using Lipofectamine 3000 reagent. Stem loop RT-qPCR was utilized to confirm that all BioRNA/let-7-5p treatments were selectively processed to their mature let-7-5p isoforms in all HCC cell lines. Western blot analysis revealed that all BioRNA/let-7-5p isoforms similarly downregulated protein level of oncogene Lin28B, with BioRNA/let-7d-5p demonstrating the strongest and most consistent downregulation across all HCC cell lines. Similarly, all HCC cell lines treated with BioRNA/let-7-5p agents also demonstrated lower ABCC5 protein level with small differences between individual isoforms. In contrast, little to no effect on the protein levels of transporters ABCC1-4 was observed following treatment with individual BioRNA/let-7-5p isoforms. These results illustrate the differences of let-7-5p family isoforms in the regulation of target gene expression in HCC cells, providing insight into how miRNA level, target complementarity, and regulatory strength may affect the anticancer potency of individual miRNA isoforms. Additionally, these results demonstrating downregulation of ABCC5 following BioRNA/let-7-5p treatment will inform the direction of future study exploring rational combination with chemotherapies in HCC. This work was supported by the National Institutes of Health National Institute of General Medical Sciences [R35-GM140835] and National Cancer Institute [R01-CA253230 and R01-CA225958]. J.M.C. was supported by a National Institutes of General Medical Sciences-funded Pharmacology Training Program grant [T32-GM144303]