Abstract 3963: Antisense agents and RNA mimics for miR-17-5p guide strand and miR-17-3p passenger strand differentiate the strength of guide and passenger strand targets in PDCD4 and PTEN mRNA 3′UTRs in MDA-MB-231 triple negative breast cancer cells

Abstract

Abstract Conventional wisdom holds that only one of the two strands in a microRNA (miRNA) precursor duplex is selected as the active guide strand. The complementary passenger strand is thought to be inactive. In triple negative breast cancer (TNBC), high levels of the miRNA guide strand called miR-17-5p inhibits ribosomal translation of tumor suppressor genes, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, we found that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human MDA-MB-231 TNBC cells, instead of raising them. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, we began seeking an answer to the contradiction by in silico analysis. We found 4 putative binding sites for miR-17-3p passenger strand in PDCD4 mRNA, and 6 in PTEN mRNA, but only one in either mRNA for the miR-17-5p guide strand. We investigated the possibility that gene targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. As a result, antisense knockdown of miR-17-3p raised PDCD4 mRNA by 25±2% and PTEN mRNA by 22±6%. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells reduced PDCD4 and PTEN protein levels. From these results, we speculated that, similar to miR-17-5p guide strand, miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked miR-17-3p passenger strand, effectively raising the miR-17-3p concentration in TNBC cells. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand. To challenge our new hypothesis, luciferase assays using reporter constructs harboring miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs are underway. No conflicts of interest. Supported by NIH CA148565 to E.W. Note: This abstract was not presented at the meeting. Citation Format: Yuan-Yuan Jin, Nicole L. Simone, Eric Wickstrom. Antisense agents and RNA mimics for miR-17-5p guide strand and miR-17-3p passenger strand differentiate the strength of guide and passenger strand targets in PDCD4 and PTEN mRNA 3′UTRs in MDA-MB-231 triple negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3963. doi:10.1158/1538-7445.AM2015-3963