A useful method of identifying of miRNAs which can down-regulate Zeb-2.
BACKGROUND
Although identification of the target mRNAs of micro RNAs (miRNAs) is essential to understanding their function, the low complementarity between miRNAs and their target mRNAs has complicated this process. In this study, we sought to identify miRNAs which reduce the expression of the transcription factor Zeb-2, a transcriptional repressor of E-cadherin which is known to be down regulated by members of the miR-200 family (miR-200a,b,c miR-429, and miR-141).


RESULTS
We first used a computational target predicting system to identify 82 candidate miRNAs which bound the 3'UTR region of the Zeb-2 mRNA. Of these 82 miRNAs, precursors for 51 were available in our miRNA precursor library. Pre-miR™ Precursor Molecules for these 51 miRNAs were co-transfected into NIH3T3 cells with a luciferase reporter vector containing the 3'UTR region of the Zeb-2 mRNA. Seven miRNAs (miR-141, mi-183, miR-200a, miR-200b, miR-200c, miR-429 and miR-666-5p) were shown to down-regulate luciferase activity and Western blotting analysis confirmed that Pre-miR™ Precursor Molecules for these seven miRNAs induced expression of E-cadherin and miScript target protector against miR-183 and miR-666-5p abrogated this effect. Moreover, an Anti-miR™ miRNA Inhibitor targeting miR-183 and miR-666-5p repressed expression of E-cadherin.


CONCLUSIONS
We have established a method to identify miRNAs that bind the 3'UTR region of the Zeb-2 mRNA and that induce expression of E-cadherin, possibly by down-regulating the expression of Zeb-2. Our method may be more widely applicable for identifying miRNAs that bind target mRNA 3'UTR regions and down-regulate the expression of proteins encoded by these mRNAs.
Department of Nephrology and endocrinology, University of Tokyo School of Medicine, 113-8655, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan. sooba-npr@umin.ac.jp.
Shigeyoshi
S
Oba
Takayuki
T
Mizutani
Etsu
E
Suzuki
Hiroaki
H
Nishimatsu
Masao
M
Takahashi
Yousuke
Y
Ogawa
Kenjiro
K
Kimura
Yasunobu
Y
Hirata
Toshiro
T
Fujita
D020413:3' Untranslated Regions / Q000235:genetics*; D000465:Algorithms; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001681:Biological Assay; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D000074165:Zinc Finger E-box Binding Homeobox 2
D020413:3' Untranslated Regions; D015820:Cadherins; D018398:Homeodomain Proteins; C533449:MIRN183 microRNA, human; D035683:MicroRNAs; D012097:Repressor Proteins; C498898:SLC4A4 protein, human; D054852:Small Molecule Libraries; D027982:Sodium-Bicarbonate Symporters; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; D008156:Luciferases