Distance between miRNA responsive elements selectively enhances target specificity with reduced cellular toxicity by miRNA-mediated synergism

Abstract

ABSTRACTDespite the emergence of miRNAs as a promising therapeutic tool in cancer management, most of the clinical trials were not successful due to their cytotoxic effects. Here, we investigated the synergistic effect of miRNA-pairs on enhancing target specificity and reducing cellular toxicity by minimizing off-target effects. Synergistic reductions by the miRNA-pairs were observed only for the common target genes having 3’-UTRs with miRNA responsive elements (MREs) within a distance between 200-800bp. Deletions of either of the miRNA seed sequence resulted in a loss of synergism. Furthermore, to study the miRNA mediated synergism in cancer cells, we cotransfected oral cancer cells with reduced doses of miRNA-pairs and determined the expressions of their common and unique target genes at both gene and protein levels. Over expressions of miRNA-pairs significantly reduced cell proliferation, migration, and spheroid formation in cancer cells compared to those with the single miRNA. Proteomic data revealed that the let-7c-5p and miR-125b-5p miRNA-mimic pairs synergistically reduced the expressions of their common target genes, hexokinase 2 (HK2) and branched chain amino acid transaminase 1 (BCAT1). The unique target genes did not exhibit any significant synergistic down-regulation in their protein levels. Metabolomic analysis consistently reflected the effect of HK2 and BCAT1 downregulation by affecting the glycolysis and the BCAA degradation pathways, suggesting the importance of this strategy in enhancing the efficacy of miRNA-mimics in cancer therapy.