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. 2023 Jan 6;12(2):233. doi: 10.3390/cells12020233

Figure 5.

Figure 5

miR-129-5p targets Kif5b mRNA and regulates Kif5b protein levels. (A) Real-time PCR analysis of normalized miR-129-5p and Kif5b mRNA expression following 24 h of miRNA overexpression (OE) in the SH-S5Y5 neuroblastoma cell line, relative to control plasmid OE. Unpaired students’ t-test. ** p < 0.01, *** p < 0.0001. (B) Sequences of Renilla/Firefly luciferase under the regulation of Kif5b 3′UTRs that were used for transient reporter assay experiments. Wild-type (WT) and mutant (mut) alleles for miR-129-5p binding sites are presented. The miRNA seed region and complimentary 3′ UTR sequence are marked in bold. Mutagenic nucleotides are marked in red and bold. (C) Luciferase activity 48 h following co-transfection of miR-129-5p with Renilla/Firefly luciferase constructs under the regulation of Kif5b 3′UTR. The graph shows the relative expression level of Firefly luciferase standardized to Renilla luciferase. The paired two-tail Student’s t-test was used for statistical analysis (n = 3, ** p < 0.01). (D,E) Western blot (D) and analysis of Kif5b protein (~110 kDa) levels in NSC-34 overexpressing miR-129-5p or scrambled control for 72 h. (E). ERK1/2 (42–44 kDa) was used as a loading control. ** p < 0.01 student’s t-test, n = 5 independent experiments.