Figure 5: CARL can regulate miR-539 expression and activity.

(a) LncRNAs expression levels upon treatment with anoxia. Cardiomyocytes were untreated (control) or treated with anoxia for 24 h. LncRNAs chosen from Fantom company were analysed by qRT-PCR. Data are shown as mean±s.e.m. of four independent experiments. *P<0.05 in Student’s t-test. (b) Knockdown of CARL induces the increase of miR-539 expression levels. Cardiomyocytes were infected with adenoviral CARL-siRNA (CARL-siRNA) and its scramble form (CARL-sc). miR-539 levels were analysed by northern blot (upper panel) and CARL levels were analysed by real-time PCR (low panel). Data are shown as mean±s.e.m. of three independent experiments. *P<0.05 versus control in one-way analysis of variance. (c) Knockdown of CARL induces miR-539 activity. Cardiomyocytes were infected with adenoviral CARL-siRNA and its scramble form, then transfected with miR-539 sensor. Luciferase activity was analysed. Data are shown as mean±s.e.m. of three independent experiments. *P<0.05 versus miR-539 sensor alone in one-way analysis of variance. (d) Enforced expression of CARL reduces the expression levels of miR-539. Cardiomyocytes were infected with adenoviral CARL or β-gal. miR-539 levels were analysed by northern blot (upper panel) and CARL levels were analysed by real-time PCR (low panel). Data are shown as mean±s.e.m. of three independent experiments. *P<0.05 versus control in one-way analysis of variance. (e) CARL reduces miR-539 activity. Cardiomyocytes were infected with adenoviral CARL or β-gal, then transfected with miR-539 sensor. Luciferase activity was analysed. Data are shown as mean±s.e.m. of three independent experiments. *P<0.05 versus miR-539 sensor alone in one-way analysis of variance. (f) CARL acts as a sponge for miR-539. Cardiomyocytes were infected with adenoviral miR-539, CARL or β-gal, then transfected with miR-539 sensor. Luciferase activity was analysed. Data are shown as mean±s.e.m. of three independent experiments. *P<0.05 in one-way analysis of variance.