Abstract Body: Backgrounds: Our prior work demonstrated the upregulation of miR-200 family miRNAs specifically miR-141 & miR-200c after stroke in mice models. Moreover, we found a significant upregulation of miR-141 in stroke patient's blood samples and validated the potential of targeting miR-141 to mitigate ischemic stroke damage. In this study, we aimed to validate the upregulation of miR-200c in stroke patients plasma samples and evaluate the potential of inhibiting miR-200c alone or in combination with miR-141 to mitigate stroke damage in oxygen-glucose deprivation (OGD) in microglial BV2 cells.
Methods: Total RNA (including miRNAs) was isolated from plasma samples of stroke patients and healthy controls and miR-200c expression was assessed by qPCR. In vitro, an oxygen-glucose deprivation (OGD) model in BV2 cells was used to assess the potential of inhibition using antagomiRs of miR-200c alone and in combination with miR-141 on cell viability, cytotoxicity, apoptosis and inflammation via MTT, LDH assays, and qPCR for gene expression. TargetScan web portal was used to obtain the potential targets of miR-200c/141 and explored the molecular mechanism behind this neuroprotection.
Results: miR-200c was significantly upregulated in stroke patients compared to healthy individuals, confirming the current study's translational potential. We found a gradual increase in miR-200c expression after 1h, 2h, 3h, and 4h (>5 folds) OGD followed by 24h reperfusion (R). Inhibition of miR-200c or miR-141/200c cluster after 4h OGD/24h R showed a synergistic effect on cell viability and cytotoxicity. Inhibition of the miR-141/200c increased anti-apoptotic gene Bcl2 and reduced the pro-apoptotic (Bax) and pro-inflammatory genes (Il-1β, Il-6, and Tnf-α) in BV2 cells following OGD/R. According to TargetScan analysis, Smad2 is a direct target of miR-200c, Tgfb2 is targeted by miR-141, and Zeb1 is commonly targeted by both miR-200c and miR-141-3p; the expression of these genes significantly decreases following OGD/R. The inhibition of miR-141/200c restored the levels of Tgfβ2, Smad2 & Zeb1.
Conclusion: Cluster miR-141/200c can act as a biomarker and a prominent therapeutic target for stroke treatment. In vitro, inhibition of miR-200c alone or together with miR-141-3p protects from OGD-induced injury in microglial BV2 cells by reducing neuroinflammation and apoptosis. Overall, the Tgfβ2/Zeb1 pathway is involved in the cluster miR-141/200c mediated neuroprotection.