Abstract B41: Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity

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 previously reported that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins in human MDA-MB-231 TNBC cells, instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of the miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by the miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and the miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3′UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knockdown miR-17-5p reduced PDCD4 and PTEN protein expression, instead of raising them. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells also reduced PDCD4 and PTEN protein levels. To test the hypothesis that miR-17-3p passenger strand can also modulate the same mRNA targets as miR-17-5p, we performed luciferase assays using reporter constructs harboring predicted miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs. We found that the miR-17-5p mimic lowered the expression of luciferase vectors containing one predicted binding site from the PDCD4 or PTEN 3′UTR domains. However, the miR-17-3p mimic lowered the expression of luciferase vectors containing all four 3′UTR target sites of PDCD4, and two of the six predicted PTEN 3′UTR target sites. In addition, we used luciferase assays to test the hypothesis that antisense DNA-LNA against miR-17-5p mimicked passenger strand miR-17-3p. We found that anti-miR-17-5p DNA-LNA lowered the expression of luciferase reporter vectors containing two of the four predicted PDCD4 3′UTR target sites for miR-17-3p, and one of the six predicted PTEN 3′UTR target sites for miR-17-3p. From these results, we concluded that the miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs, just like the miR-17-5p guide strand. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked the 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. Supported by the Department of Biochemistry and Molecular Biology. Conflict of interest: pending PCT/US2015/015681 patent application licensed to Nikita RNA LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B41.