Objectives To evaluate the expression profile of myoD microRNA-29(miR-29)family in L6myoblast differentiated to myotube or L6 myotube treated by glucose and insulin,and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.Methods The expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis.The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search.The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay.Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence.Moreover,overexpression of myoD was achieved by a recombinant adenovirus system(Ad-myoD).L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.Results The expression levels of myoD,miR-29a,miR-29b,and miR-29c were increased in L6myoblast differentiated to myotube.The expression of myoD,miR-29b,and miR-29c was up-regulated in L6myotube treated with glucose and insulin,but miR-29a depicted no significant change.Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster.Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.Conclusion MyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.
Objective To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tissues and to validate its bona fide targets in human cholangiocarcinoma cells. Methods The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholangiocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respectively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarcinoma tissues by using real-time PCR, locked nucleic acid in situ hybridization (LNA-ISH), and immunohistochemistry analysis. Results Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bileduct tissues (P<0.05). The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholangiocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN (P<0.01) and PDCD4 (P<0.05) and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue. Conclusion microRNA-21 expression is up-regulated in human cholangiocarcinoma and PTEN, PDCD4 are direct effectors of microRNA-21.
