Placenta-specific miRNA (miR-512-3p ) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells

Abstract Aim The micro RNA s (mi RNA s) derived from the chromosome 19 miRNA cluster ( C19MC ) are exclusively expressed in the human placenta, but the origin and functions of C 19 MC mi RNA s are not fully understood. The purpose of this study was to elucidate which cells express C 19 MC mi RNA s 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 C 19 MC mi RNA s (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 C 19 MC mi RNA s 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 P re‐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.