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Fig. 5 | Journal of Biomedical Science

Fig. 5

From: The miR-15b-Smurf2-HSP27 axis promotes pulmonary fibrosis

Fig. 5

Involvement of miR-15b in downregulation of Smurf2. A Online prediction databases (TargetScan, miRDB, and miRSystem) were used to predict miRNAs targeting Smurf2. B miRNA expression measured by qPCR at each time point after 10 Gy IR. **P < 0.01 vs. control. C Cell lysates 24 h after transfection to mimic RNAs (100 nM). Analysis was performed by western blot and qRT-PCR; gapdh mRNA was used for normalization. *P < 0.05 vs. miR-NC. D Putative miR-15b binding site in the human Smurf2 3’-UTR and luciferase constructs with the wild-type (WT) and mutant (Mut) miR-15b target sequences. The red colors indicate the mutant sequences of the 3’-UTR (upper). L132 cells were co-transfected with 50 and 100 nM of the miR-15b together with the pmirGLO dual-luciferase vectors containing the wild type or mutant 3’-UTR of Smurf2. Data are represented as the mean ratios of Renilla to Firefly luciferase activity and are normalized relative to the negative control (bottom). **P < 0.01 and ***P < 0.001 vs. WT-NC; ###P < 0.001 vs. WT-100 nM. E Cell lysates 9 h after 8 Gy IR with or without anti-miR-15b (50 nM). Analysis was performed by western blot and qRT-PCR; gapdh mRNA was used for normalization. Ratio of each protein to β-actin in all western blot data (n ≥ 3, mean ± SD). Subsequent statistical analysis was performed with one-way ANOVA with Newman-Keuls test for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control; #P < 0.05 and ##P < 0.01 vs. IR only. F Periodic expression was confirmed by qRT-PCR using lungs from individual mice. The miR-15b expression was normalized to that of U6. The smurf2 expression was normalized to that of gapdh (n = 3, mean ± SD). *P < 0.05 and **P < 0.01 vs. control

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