Glutathione (GSH) is a key antioxidant that plays an important neuroprotective role in the brain. Decreased GSH levels are associated with neurodegenerative diseases. We previously reported that one of the important microRNA, contributed the neuroprotection against oxidative stress through regulating the expression of excitatory amino acid carrier 1 (EAAC1) and GSH levels. In this study, we focused on GTRAP3-18, the negative factor of EAAC1, as a new target of miR-96-5p.
First, we investigated whether the expression of GTRAP3-18 is affected by manipulation of miR-96-5p level using western blot analysis and luciferase reporter gene assay in human neuroblastoma SHSY-5Y cells. Next, we identified the candidates of GTRAP3-18 regulators using mass spectrometry analysis since we found out GTRAP3-18 is indirectly regulated by miR-96-5p. Then, we have tested whether these candidate proteins could be a direct regulator of GTRAP3-18.
The result shows that GTRAP3-18 is up-regulated by miR-96-5p at transcriptional and translational levels. Furthermore, we identified several miR-96-5p regulating RNA binding proteins that negatively regulate GTRAP3-18.
In conclusion, miR-96-5p could reduce RNA-binding proteins that down-regulate GTRAP3-18 to decrease neuronal GSH levels.