The phenotype observed when inhibiting an miRNA can be due to many targets being upregulated. Target site blockers (TSB) lead to the upregulation of one specific target and may or may not cause the same phenotype.
miR-199a-5p stimulates TGFbeta signaling by turning down CAV1 expression. Inhibiting miR-199a-5p directly using a miRNA inhibitor (LNA-miR-199a-5p) or inhibiting the interaction of miR-199a-5p with CAV1 using a target site blocker (CAV1 protector), reduces TGFbeta signaling and causes several phenotypes. (C) Both inhibition of miR-199a-5p or using the CAV1 target site blocker (TSB) reduces myofibroblast differentiation as visualized by alpha-smooth muscle actin (a-SMA) staining (green). (D) Co-transfection of the miR-199a-5p inhibitor as well as the CAV1 TSB reduces expression from a SMAD-luciferase reporter plasmid. (E and F) Both inhibition of miR-199a-5p or using the CAV1 TSB reduces the migration rate of TGFbeta stimulated hFL1 lung fibroblasts leading to slower wound closure. From: Cardenas CL, et al. miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1. PLoS Genet. 2013;9(2):e1003291.
(A) Target site blockers are antisense oligonucleotides designed to compete with miRNA/RISC by hybridizing to the miRNA target site of a particular mRNA. (B) LNA-enhanced, high-affinity target site blockers compete effectively with miRNA/RISC for the miRNA target site. In addition, LNA distribution throughout the LNA/DNA mixmer ensures that the antisense oligonucleotide does not catalyze RNase H-dependent degradation of the mRNA. As a result, the TSB will cause increased expression of the protein encoded by the targeted mRNA.