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- Title
Placenta-specific miRNA ( miR-512-3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells.
- Authors
Kurashina, Ryuhei; Kikuchi, Kunio; Iwaki, Jun; Yoshitake, Hiroshi; Takeshita, Toshiyuki; Takizawa, Toshihiro
- Abstract
Aim The micro RNAs (mi RNAs) derived from the chromosome 19 miRNA cluster ( C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19 MC mi RNAs are not fully understood. The purpose of this study was to elucidate which cells express C19 MC mi RNAs 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 C19 MC mi RNAs (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 C19 MC mi RNAs 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-mi R-512-3p (mature mi R-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. Conclusion These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.
- Subjects
ANALYSIS of variance; BIOLOGICAL assay; CELL culture; CHORIONIC villi; GENE expression; GENETIC techniques; HYDROLASES; PHOSPHATASES; PLACENTA; POLYMERASE chain reaction; RESEARCH funding; RNA; STATISTICS; T-test (Statistics); WESTERN immunoblotting; DATA analysis; DATA analysis software; MICROARRAY technology; DESCRIPTIVE statistics
- Publication
Journal of Obstetrics & Gynaecology Research, 2014, Vol 40, Issue 3, p650
- ISSN
1341-8076
- Publication type
Article
- DOI
10.1111/jog.12217