INTRODUCTION: MicroRNAs (miRNAs) are small noncoding RNAs that bind to target sites in mRNAs, leading to translational repression. MiRNAs are present in dendrites and synapses where they are believed to fine‑tune the local expression of synaptic proteins. MiR-132 is a neuronal activity-regulated microRNA that controls the morphology of dendritic spines and synaptic transmission. Similar activities have recently been attributed to matrix metalloproteinase-9 (MMP-9), an extrasynaptic protease. Our previous studies show that miR-132 can directly regulate Mmp-9 mRNA by targeting its 3’UTR in cultured primary neurons. AIM(S): In the current study, we aimed at verification whether miR-132 regulates the expression of Mmp-9 in vivo in the mouse brain. METHOD(S): To determine whether miR-132 binds to the 3’UTR of Mmp-9 mRNA, the luciferase reporter assay using the coding sequence of firefly luciferase fused with the 3’UTR of Mmp-9 mRNA. Next, CRISP-Cas9 technology was used, in order to introduce mutations in putative binding site for miR-132 in 3’UTR of Mmp-9 locus in mice. Subsequently, gelatin zymography was used to evaluate the levels of MMP‑9 protein in different brain regions of mutant and control mice. RESULTS: Overexpression of miR-132 in cortical neurons significantly reduced the luciferase activity of MMP‑9 3’UTR reporter. Importantly, miR-132 failed to regulate the mutated MMP‑9 3’UTR luciferase reporter, confirming the functionality of the predicted sequence within the 3’UTR of MMP-9. Mutation in 3’UTR region of MMP-9 targeted by miR-132 in mice, resulted in higher MMP-9 protein levels in different brain regions of mutant mice as compared to controls. CONCLUSIONS: We show, that miR-132 binds to the 3’UTR of Mmp-9 mRNA in primary cortical neurons. Moreover, we developed a new mouse model to study miR-132 – Mmp-9 interaction in vivo. Our data suggest, that miR-132 targets 3’UTR of Mmp-9 mRNA in vivo and can regulate MMP-9 protein in mouse brain. FINANCIAL SUPPORT: Supported by NCN OPUS 2014/15/B/NZ3/01054.
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