Abstract DP270: Analysis of Cerebro Protective Role of miR-141/200c Inhibition/Deletion in Mouse Model of Ischemic Stroke

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. Mirc13^tm1Mtm/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.