Research Paper Advance Articles pp 23974—23995

Effect of lncRNA WT1-AS regulating WT1 on oxidative stress injury and apoptosis of neurons in Alzheimer's disease via inhibition of the miR-375/SIX4 axis

Figure 5. miR-375 promoted OSI and apoptosis by inhibiting the expression of SIX4. (A) Prediction of the target genes of miR-375, with the middle part representing the intersection of target prediction results and downregulated genes in GSE4757. (B) Differential expression of candidate target genes in GSE4757 (*p<0.05, **p<0.01). (C) The expression of SIX4 detected by qRT-PCR in Aβ25-35-treated SH-SY5Y cells. (D) Bioinformatic analysis predicted the presence of a binding site between miR-375 and SIX4 mRNA. (E) Dual luciferase reporter assay showed that miR-375 directly bound SIX4 mRNA. (F) RIP assay proved that miR-375 directly bound to SIX4. (G) The mRNA and protein expression of SIX4 after overexpression of miR-375 detected by qRT-PCR and western blot. (H) The expression of miR-375 and SIX4 detected by qRT-PCR. (I) The expression of p-Tau and total Tau detected by western blot. (J) Detection of mitochondrial membrane potential by JC-1 staining. (K) Detection of ATP content. (L) Detection of ROS content. (M) Detection of MDA content, SOD activity and GSH-Px activity. (N) Detection of LDH activity. (O) Detection of apoptosis by flow cytometry. *P<0.05; the experimental results are expressed as the mean ± standard deviation. Differences among multiple groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test. The experiment was repeated three times.