Real-time functional imaging for monitoring miR-133 during myogenic differentiation.
MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events.
Kato
Yoshio
Y
Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba 305-8562, Japan. y-kato@aist.go.jp
Miyaki
Shigeru
S
Yokoyama
Shigetoshi
S
Omori
Shin
S
Inoue
Atsushi
A
Horiuchi
Machiko
M
Asahara
Hiroshi
H
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics*; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000235:genetics*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D035683:MicroRNAs; D000097573:Red Fluorescent Protein; C517732:Mirn133 microRNA, mouse