Fig. 7

Clu mRNA is a biological target of miR-22-5p. (a) 3’UTR sequence of Clu with indicated miR-22-5p target binding sequence (miRDB database). (b) MiR-22-5p-Clu target binding validation assay. Relative luciferase activity was measured in HEK293T cells co-transfected with pmirGLO Dual-Luciferase expression vector containing an approximately 70 bp-long sequence of 3′ UTR Clu and with miR-22-5p mimics or NC. Data are representative of three independent experiments and expressed as the mean ± SEM. Data were analyzed using unpaired t-test, p = 0.0008. (c) Quantification of miR-22-5p expression in astrocytes transfected with miR-22-5p mimic or NC. Cells treated with the transfectant agent NM were used as additional control. miR-22-5p expression was normalized on the geometric mean of the reference RNAs and expressed as rq. Data are representative of three independent experiments and expressed as the mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test; NC vs. mimic miR-22-5p, p < 0.0001; NM vs. mimic miR-22-5p, p < 0.0001. (d) Quantification of Clu gene expression in astrocytes transfected with miR-22-5p mimic or NC. Cells treated with NM were used as additional control. Clu gene expression was normalized on the geometric mean of the HK genes and expressed as rq. Data are representative of three independent experiments and expressed as the mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. (e) Quantification of Clu protein in astrocytes transfected with miR-22-5p mimic or NC. Cells treated with NM were used as additional control. preClu and the cleaved form were normalized on GAPDH. Data are representative of three independent experiments validated on three different primary cultures and expressed as the mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test; preClu: NC vs. mimic miR-22-5p, p = 0.0007; NM vs. mimic miR-22-5p, p = 0.0014. Clu: NC vs. mimic miR-22-5p, p = 0.0006; NM vs. mimic miR-22-5p, p = 0.0015