Fig. 3 Target threshold in relief from miRNA mediated repression using molecular titration model . The model assumes that the target mRNA can get produced and degraded intrinsically. After interacting with miRNA, mRNA gets degraded or dissociated from the complex, while the miRNA is recycled back into the system. The model also assumes that the protein translation can occur only from the free mRNA and the mRNA bound to the miRNA cannot undergo translation. Plots of target gene expression by varying, (a) the number of miRNA binding sites on the target, and (b) the concentration of the miRNA mimics for the same target with ‘n’ number of binding sites. Violet dotted lines represent the target expression in the absence of a miRNA binding site. Adapted from Mukherji et al53.
Gam et al54 investigated the fold repression using a similar dual fluorescence reporter but with a miRNA target site consisting of four perfectly complementary repeat sequences for the miRNA in the 3’UTR of the fluorescent gene.54 They created a library of miRNA sensors for various miRNAs and observed the threshold kind of behavior similar to that of Mukherji et al53 for a single miRNA binding site (four repeat sequences) with the perfect match. They found that there are three output regimes for miRNA mediated gene expression: (i) mRNA repressed regime (ii) mRNA derepressed regime and (iii) an in-between threshold regime (where switching from mRNA repression to de-repression takes place). They have demonstrated that the number of binding sites and miRNA levels can shift the threshold for miRNA-mediated mRNA repression.54 High throughput assays performed to measure miRNA activity for a large number of miRNAs in other cell lines further suggest that the factors such as miRNA abundance and no. of binding sites highly influence the miRNA repression activity.52
Such sharp threshold response is observed for Notch (a signaling molecule) expression for color cancer stem cells (CCSCs), where mir-34a creates a bimodality in Notch expression by sequestering Notch mRNA.63 The Notch pathway plays an important role in regulating asymmetric division in both normal and cancer stem cells, thus creating a CCSC daughter cell with self-renewal potential and a differentiated non-CCSC daughter cell. It has been observed that injecting these cells into the mouse xenograft models, low miR-34a expressing CCSCs promoted tumor growth due to symmetric division of CCSCs to give two CCSC daughter cells. On the other hand, high mir-34 expressing cells promoted differentiation with reduced tumorigenicity. Hence, the ability of microRNAs to fine-tune the symmetric and asymmetric division of cancer stem cells can be used as a potential strategy for cancer treatment.63