Placenta-specific miRNA (miR-512-3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells.
OBJECTIVE
The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets.


METHODS
A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay.


RESULTS
By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system.


CONCLUSIONS
These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.
Department of Molecular Medicine and Anatomy, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan; Department of Reproductive Medicine, Perinatology and Gynecologic Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
Ryuhei
R
Kurashina
Kunio
K
Kikuchi
Jun
J
Iwaki
Hiroshi
H
Yoshitake
Toshiyuki
T
Takeshita
Toshihiro
T
Takizawa
D000328:Adult; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D005260:Female; D006801:Humans; D061346:Laser Capture Microdissection; D035683:MicroRNAs / Q000378:metabolism*; D009928:Organ Specificity; D010920:Placenta / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism
C000595430:MIRN512 microRNA, human; D035683:MicroRNAs; D011994:Recombinant Proteins; D019703:Calcineurin; C525676:PPP3R1 protein, human
PPP3R1; calcineurin; human placenta; miR-512-3p; miRNA