Background: Our previous work demonstrated the expression of miR-141 and miR-200c—grouped as the genomic cluster miR-141/200c (chr12p13. 31 in humans) —is elevated after ischemic stroke, both in experimental models and in stroke patients. Recently, we validated the therapeutic potential of miR-141 inhibition to mitigate ischemic damage. The present study evaluates the functional relevance and molecular mechanisms of the miR-141/200c cluster in the ischemic brain. Methods: A middle cerebral artery occlusion (MCAo) model was used to induce stroke in C57BL/6 mice. Cell-specific localization of the candidate miRNAs was assessed using RNA fluorescence in situ hybridization (RNA-FISH) and quantified by RT-qPCR following flow cytometry-based cell sorting of major brain cells. An in vitro oxygen-glucose deprivation (OGD) model was established in BV2 microglial cells and effects of miR-141/200c inhibition on cell viability, cytotoxicity, apoptosis, and inflammation assessed by MTT, LDH, TUNEL assays, and qPCR. Mirc13ᵗm1Mtm/Mmjax (MMRRC Strain #034657-JAX) mice were used to generate first conditional, then global and cell-specific KO lines using Cre-expressing mouse strains as provider’s protocol. miR-141/200c expressions were further confirmed in KO and littermate wild-type (WT) mice using RNA-FISH and RT-qPCR. Global KO mice were subjected to 1-hour MCAo followed by 24 hours of reperfusion (R). Infarct volume and neurological deficits (ND) were assessed by TTC staining and ND scoring. Results: RNA-FISH analysis revealed expression of miR-141 and miR-200c in neurons, astrocytes, and microglia, with highest microglial upregulation after stroke (in flow-sorted cells). In BV2 cells, the inhibition of miR-141/200c after 4 hours of OGD/R significantly increased cell viability, reduced cytotoxicity, and decreased expression of pro-apoptotic (Bax) and pro-inflammatory genes (Il-1β&Tnf-α). Additionally, combined inhibition of miRNAs restored expression of Tgfβ pathway genes (Tgfβ2, Zeb1, Smad2/3). Consistently, in vivo data from KO mice demonstrated a significant post-stroke infarct volume reduction (~40%) and improved neurological deficits (P<0. 05 vs. WT). Conclusion: Inhibition of the miR-141/200c cluster reduces infarct injury by upregulating Tgfβ-Zeb1 pathway proteins, suggesting a promising therapeutic target for improving ischemic stroke recovery. Future studies using these novel KO models will help delineate the detailed mechanisms underlying this neuroprotection.
Yadav et al. (Thu,) studied this question.