Research Paper Advance Articles pp 24033—24056

lncRNA DLEU2 acts as a miR-181a sponge to regulate SEPP1 and inhibit skeletal muscle differentiation and regeneration

Figure 6. (A) Bioinformatics prediction of miR-181a as the target miRNA of DLEU2 using RNAhybrid 2.12. MFE: Minimum free energy. (B) C2C12 cells were transfected with different doses of biotin-labeled DLEU2. Results of pull-down experiments for miR-181a and real-time PCR assay results are shown. And biotinylated DLEU2 pulled down some miR-181a in C2C12 cells. * P < 0.05 vs 0.5 mM. (C) Determination of miR-181a regulation by DLEU2 by Luciferase reporter assays. * P < 0.05 vs miR-NC. (D,E) Real-time PCR and Western blot results showing mRNA and protein expression of SEPP1, MyoD and MyoG in C2C12 cells co-transfected with mir-181a mimic or mir-181a inhibitor following DLEU2 overexpression. Data are presented as mean ± SD. U6 small nuclear RNA served as the internal control for lncRNA and miRNA. GAPDH mRNA was used as the control mRNA. (F) Effect of DLEU2 overexpression on the proliferation of C2C12 cells. Impact of miR-181a inhibitor and miR-181a mimic of cell proliferation. (G) Cells transfected with DLEU2 and treated with the miR-181a inhibitor showed a significant decrease in the level of proliferation, whereas cells treated with the miR-181a mimic showed increased proliferation. Quantification of relative ratio of Edu+ C2C12 cells. Data are shown as the mean ± S.D. (n = 3). *** p < 0.005, **** p < 0.0005.