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. 2018 Nov 6;7(11):199. doi: 10.3390/cells7110199

Figure 3.

Figure 3

CircFGFR2 sponges with miR-133a-5p and miR-29b-1-5p, and inhibits the expression of miR-133a-5p and miR-29b-1-5p in myoblast. (A–C) The potential binding sites of miR-133a-5p and miR-29b-1-5p in circFGFR2. The mutant sequences in binding sites are highlighted in red. (D) A schematic drawing showing the putative binding sites of miR-133a-5p/miR-29b-1-5p associated with circFGFR2. (E,F) Luciferase assay was conducted by co-transfecting wild type or mutant linear sequence of circFGFR2 with miR-133a-5p/miR-29b-1-5p mimic or mimic-NC in DF-1 cells. (G,H) Luciferase assay was conducted by co-transfecting wild type circFGFR2 linear sequence and miR-133a-5p/miR-29b-1-5p mimic or mimic-NC and with circFGFR2 overexpression vector (pCD2.1-circFGFR2) or empty vector (pCD2.1-ciR). (I) Biotin-coupled miRNA pull down assay from the myoblast lysates after transfection with 3′ end biotinylated miR-133a-5p, miR-29b-1-5p or mimic NC. The expression level of circFGFR2 was quantified by qRT–PCR, and fold enrichment in the streptavidin captured fractions are plotted. (J,K) qRT–PCR analysis of the relative expression of miR-133a-5p and miR-29b-1-5p after overexpression or inhibition of circFGFR2. In all panels, results are expressed as the mean ± S.E.M. of three independent experiments. For two group comparison analysis, statistical significance of differences between means was analyzed by unpaired Student’s t-test. For multiple comparison analysis, data were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p < 0.05 to be statistically significant. * p < 0.05; ** p < 0.01. NC, negative control.