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

Despite the emergence of miRNAs as promising therapeutic tools in cancer management, most clinical trials have not been successful due to their cytotoxic effects. Here, we have investigated the factors regulating the effect of miRNA-mimic pairs in enhancing target gene specificity while reducing cellular toxicity. Synergistic reductions by the miRNA-mimic pairs were observed for the 3-UTRs of common target genes with miRNA-responsive elements (MREs) preferentially located at a distance of 200-800bp. Deletion of either of the miRNA seed sequences resulted in a loss of synergism. Furthermore, we performed small RNA-sequencing to identify significantly downregulated miRNAs in Oral cancer. Consequently, we transfected let-7c-5p and miR-125b-5p miRNA mimics either alone or in combination at half-dose concentrations and determined the expression levels of their common and unique target genes in oral cancer cells. Significant reductions in target gene expression were observed for genes containing MREs for both miRNA mimics within the preferential distance. Proteomic data revealed that the let-7c-5p and miR-125b-5p miRNA-mimic pairs synergistically reduced the expression of their common target genes, hexokinase 2 (HK2) and branched-chain amino acid transaminase 1 (BCAT1), in cancer cells. However, unique target genes did not exhibit any significant alterations in their protein levels. The effect of HK2 and BCAT1 downregulation was also reflected in the metabolomic profiling of cancer cells, specifically affecting glycolysis and the BCAA degradation pathways. As a result of the metabolic impairment caused by the synergistic effect of miRNA mimic pairs, oral cancer cells showed a significant reduction in proliferation, migration, and spheroid formation compared to cells treated with either single miRNA. The synergistic effect of these miRNA mimics was lost in normal keratinocytes, possibly due to low expression levels of the target oncogenes, suggesting a cancer cell-specific effect of this mechanism.