Recombinant miR-7-5p Effectively Inhibits NSCLC Cell Viability through Regulating Mitochondrial Function

Abstract

<b>Abstract ID 15136</b> <b>Poster Board 245</b> Non-Small Cell Lung Cancer (NSCLC) makes up 84% of all lung cancer cases and lack of effective treatment options contribute to low survival rates. RNA interfering (RNAi) microRNA (miRNA or miR) provides researchers with a versatile alternative to modulate target gene expression to combat disease. Of these, the level of tumor suppressive miR-7-5p (miR-7) is reduced in NSCLC. Notably, miR-7 downregulates many genes important in metabolism and mitochondrial function including EGFR and VDAC1, and it is a putative regulator of mitochondrial genes AGK, PPIF, and DTYMK as well as the SLC25A15 transporter. In previous studies, reintroduction of miR-7-5p has been reported to inhibit NSCLC cell growth and metastasis, however the miRNA mimics used in those studies are chemically synthesized in&nbsp;vitro and comprised of extensive modifications that may trigger immunogenic and off-target responses. By contrast, our laboratory has developed a novel technology to bioengineer RNAi agents (BioRNA) in&nbsp;vivo utilizing specific tRNA/pre-miRNA carriers to preserve the natural post-transcriptional modifications, folding, and functions of genome derived miRNAs. Using this technology, we aim to develop BioRNA/miR-7 as a novel, bio-synthesized anticancer agent that functions through endogenous RNAi mechanisms to reduce cancer cell viability through the regulation of mitochondrial function. Thus far, we have successfully cloned and overexpressed leucyl and glycyl tRNA versions of BioRNA/miR-7 in Escherichia coli as verified by Sanger sequencing and RNA gel electrophoresis, respectively. We have isolated leucyl and glycyl BioRNA/miR-7 from total bacterial RNA by anion exchange Fast Protein Liquid Chromatography to attain a purity of 99.5% and 98.7%, respectively, as determined by reverse phase High Performance Liquid Chromatography. Additionally, endotoxin assay showed low endotoxin levels of 0.74 and 0.68 (EU/μg RNA) for leucyl and glycyl BioRNA/miR-7, respectively. Moreover, we have established the effects of BioRNA/miR-7 to reduce cell viability via IncuCyte Live Cell Image Analyzer, validated the processing of glycyl tRNA-BioRNA/miR-7 to target miR-7-5p in NSCLC cells by stem-loop reverse transcription real-time qPCR assay, and assessed the functional capability of BioRNA/miR-7 to regulate known miR-7 target gene expression in comparison to a commercial miR-7 mimic by Western blot and immunofluorescent microscopy. Furthermore, we have established the effects of BioRNA/miR-7 on mitochondrial function via Seahorse Analyzer and compared treatment-induced changes in mitochondrial morphology by confocal microscopy. The success of the BioRNA technology together with previous and preliminary data on miR-7 suggests that miR-7 plays an important role in mitochondrial function, and BioRNA/miR-7 may be developed as a new therapeutic RNA for the treatment of NSCLC. Supported by the National Institute of General Medical Sciences [R35GM140835] and National Cancer Institute [R01CA225958 and R01CA253230], National Institutes of Health. Gavin M. Traber is supported by a National Institutes of General Medical Sciences-funded Pharmacology Training Program Grant [T32GM099608 and T32GM144303].