Background: We intended to examine the functional role of microRNA 26 (miR-26a) in regulating H2O2-induced cytotoxicity and apoptosis in RGC-5 cells in vitro.
Method: Various concentrations of H2O2 (0-1000 μM) were added in RGC-5 culture. Cell cytotoxicity was monitored by viability assay and gene expression level of miR-26a examined by qRT-PCR. MicroRNA-26a mimic was then applied in the RGC-5 culture to examine its effect on upregulating endogenous miR-26a and rescuing H2O2-induced cytotoxicity. TUNEL immunostaining assay was used to further assess the protective effect of upregulating miR-26a on H2O2-induced apoptosis in RGC-5 cells. Direct targeting of miR-26a on Phosphatase and tensin homolog (PTEN) signaling pathway was assessed by luciferase assay and western blotting. PTEN was then ectopically over-expressed in RGC-5. And its effects on miR-26a mediated apoptosis protection in RGC-5 were investigated by western blot and TUNEL assay.
Results: H2O2 induced cytotoxicity and down-regulated miR-26a in dose-dependent manner in RGC-5 cells. MiR-26a-mimic upregulated endogenous miR-26a gene levels, and then reduced H2O2-induced cytotoxicity, as well as H2O2-induced apoptosis in RGC-5 cells. PTEN was directly targeted by miR-26a. PTEN protein was upregulated, and phosphorylated AKT protein down-regulated while miR-26a was upregulated to reduce H2O2-induced apoptosis. Finally, overexpressing PTEN reversed the protective effect of miR-26a upregulation on RGC-5 apoptosis.
Conclusion: Upregulating miR-26a protects RGC-5 cell against cytotoxicity and apoptosis, probably through down-regulation of PTEN.
Keywords: Apoptosis; Neuroprotection; PTEN; RGC-5; miR-26a.
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