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. 2018 Oct 25;26(11):2669–2680. doi: 10.1016/j.ymthe.2018.09.014

Figure 3.

Figure 3

Validation of the Binding of lncMUMA with miR-762 and miR-762 with MyoD in C2C12 Cells In Vitro

(A) Bioinformatic prediction of miR-762 as a target miRNA of lncMUMA by RNAhybrid 2.12. mfe, minimum free energy. (B) Pull-down assay combined with real-time PCR analysis of miR-762 level in C2C12 cells transfected with biotin-labeled lncMUMA at different dosages. *p < 0.05 versus 0.5 mM, #p < 0.05 versus 5 mM. (C) Sequence of wild-type and mutated binding site between miR-762 and lncMUMA. (D) Luciferase reporter assay of either wild-type miR-762-transfected (miR-762-WT) or mutated miR-762-transfected (miR-762-Mut) C2C12 cells treated with negative control (lncMUMA-NC), wild-type binding site of lncMUMA (lncMUMA-WT), and mutated binding site of lncMUMA (lncMUMA-Mut), respectively. *p < 0.05 versus lncMUMA-NC. (E) Western blot analysis of expression level of MyoD in C2C12 cells treated with either agomiR-762 or antagomiR-762. *p < 0.05 versus control, #p < 0.05 versus agomiR-762. (F) Sequence of wild-type and mutated binding site between miR-762 and 3′ UTR of MyoD. (G) Luciferase reporter assay of MyoD 3′ UTR and MyoD 3′ UTR-Mut in C2C12 cells transfected with either miR-762-WT or agomiR-762-Mut. *p < 0.05 versus agomiR-762-NC. (H) Luciferase reporter assay of MyoD 3′ UTR in C2C12 cells treated with antagomiR-762. *p < 0.05 versus control. n = 5 for each group. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. β-actin is used as the internal control for protein. Data are presented as mean ± SEM.