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Supplementary Material for: MicroRNA-128 Protects Dopamine Neurons from Apoptosis and Upregulates the Expression of Excitatory Amino Acid Transporter 4 in Parkinson’s Disease by Binding to AXIN1

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posted on 07.12.2018 by Zhou L., Yang L., Li Y., Mei R., Yu H., Gong Y., Du M., Wang F.
Background/Aims: Parkinson’s disease (PD) is a frequently occurring condition that resulted from the loss of midbrain neurons, which synthesize the neurotransmitter dopamine. In this study, we established mouse models of PD to investigate the expression of microRNA-128 (miR-128) and mechanism through which it affects apoptosis of dopamine (DA) neurons and the expression of excitatory amino acid transporter 4 (EAAT4) via binding to axis inhibition protein 1 (AXIN1). Methods: Gene expression microarray analysis was performed to screen differentially expressed miRNAs that are associated with PD. The targeting relationship between miR-128 and AXIN1 was verified via a bioinformatics prediction and dual-luciferase reporter gene assay. After separation, DA neurons were subjected to a series of inhibitors, activators and shRNAs to validate the mechanisms of miR-128 in controlling of AXIN1 in PD. Positive protein expression of AXIN1 and EAAT4 in DA neurons was determined using immunocytochemistry. miR-128 expression and the mRNA and protein levels of AXIN1 and EAAT4 were evaluated via RT-qPCR and Western blot analysis, respectively. DA neuron apoptosis was evaluated using TUNEL staining. Results: We identified AXIN1 as an upregulated gene in PD based on the microarray data of GSE7621. AXIN1 was targeted and negatively mediated by miR-128. In the DA neurons, upregulated miR-128 expression or sh-AXIN1 increased the positive expression rate of EAAT4 together with mRNA and protein levels, but decreased the mRNA and protein levels of AXIN1, apoptosis rate along with the positive expression rate of AXIN1; however, the opposite trend was found in response to transfection with miR-128 inhibitors. Conclusion: Evidence from experimental models revealed that miR-128 might reduce apoptosis of DA neurons while increasing the expression of EAAT4 which might be related to the downregulation of AXIN1. Thus, miR-128 may serve as a potential target for the treatment of PD.

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