Research Paper Volume 16, Issue 1 pp 299—321

Figure 2. miR-377-3p inhibits the expression of Nampt in astrocytes after OGD/R, thus promoting neuronal injury. (A) Possible miRNA binding site on Nampt mRNA (predicted candidate miR-377-3p target gene by bioinformatics databases). (B) GFAP immunofluorescence validation of astrocytes (immunofluorescence images of primary astrocytes. Scale bar: 20 μm). (C) Relative luciferase activity of Nampt wild-type and 3ʹ-UTR mutant structures transfected with miR-377-3p mimics and NC mimic. (D) RT-qPCR detection of miR-377-3p levels in primary astrocytes after OGD/R following miR-377-3p mimic or miR-377-3p inhibitor treatment. (E) Western blot analysis of Nampt protein expression in primary astrocytes after OGD/R following miR-377-3p mimic or miR-377-3p inhibitor treatment. (F) A bar presenting the quantification of Nampt in primary astrocytes. (G) NeuN immunofluorescence validation of neurons (immunofluorescence images of primary neurons. (Scale bar: 20 μm.). (H) The co-cultured neuron viability was determined by CCK-8 assay after miR-377-3p mimic or miR-377-3p inhibitor transfection in astrocytes and OGD/R treatment. (I) LDH assay to detect the effect on co-cultured neurons after miR-377-3p mimic or miR-377-3p inhibitor treatment in primary astrocytes and OGD/R treatment. The relative expression levels were quantified by normalizing to β-actin. Data are represented as mean ± SD, (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001).