Expression of microRNA-29b2-c cluster is positively regulated by MyoD in L6 cells.
OBJECTIVE
To evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of 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 L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube 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.


CONCLUSIONS
MyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.
Department of Biochemistry, National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.
Chang-zheng
CZ
Liu
Jing-jing
JJ
Li
Jin-mei
JM
Su
Tao
T
Jiao
Li-juan
LJ
Gou
Xiao-dong
XD
He
Yong-sheng
YS
Chang
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D005810:Multigene Family / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D013549:Sweetening Agents / Q000494:pharmacology
D007004:Hypoglycemic Agents; D007328:Insulin; C547014:MIRN29 microRNA, mouse; D035683:MicroRNAs; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D013549:Sweetening Agents; D005947:Glucose