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. 2019 Feb 14;23(5):3302–3316. doi: 10.1111/jcmm.14220

Figure 6.

Figure 6

Ccnb1, the gene for cyclin B1, is the target gene of miR‐181c‐5p in primary mouse osteoblasts. miRNA target prediction tools were used to screen for cyclin B1‐targeting miRNAs, and the top ten miRNAs that received the highest composite score were selected for the expression assay. A, qPCR analysis of changes in expression of miR‐181a‐5p, miR‐181b‐5p, miR‐181c‐5p, miR‐181d‐5p, miR‐1942, miR‐6388, miR‐1954, miR‐3089‐3p, miR‐300‐5p and miR‐411‐3p in osteoblasts treated with simulated microgravity (n = 6). B, A schematic illustration of the design of luciferase reporters containing the WT Ccnb1 3′UTR (WT 3′UTR) or the site‐directed mutant Ccnb1 3′UTR (MUT 3′UTR). Sequences below indicate putative miR‐181c‐5p target sites on the WT 3′UTR, the MUT derivative, and the pairing regions of miR‐181c‐5p. C, The effects of the miR‐181c‐5p mimic and inhibitor or their negative controls on the luciferase activity of the WT Ccnb1 3′UTR or MUT Ccnb1 3′UTR reporter in 2 T3 cells. The values in the condition of WT Ccnb1 3′UTR or MUT Ccnb1 3′UTR are shown relative to that of the mimic‐NC in the same condition (n = 3). D, qPCR of miR‐181c‐5p levels in osteoblasts after treatment with mimic‐181c‐5p, inhibitor‐181c‐5p or their negative controls (n = 3). E, qPCR experiments were performed to detect changes in Ccnb1 mRNA expression in osteoblasts after treatment with mimic‐181c‐5p, inhibitor‐181c‐5p or the negative controls (n = 3). F, Western blot analyses of cyclin B1 proteins levels in primary mouse osteoblasts after treatment with mimic‐181c‐5p, inhibitor‐181c‐5p or the negative controls for 48 h. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). The histogram illustrated the relative expression of cyclin B1 present in cells from each group as quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 3). The results were expressed as the mean ± SD with a one‐way ANOVA with a SNK‐q test. *P < 0.05 and **P < 0.01, compared with the stationary control.