Forskolin-mediated BeWo cell fusion involves down-regulation of miR-92a-1-5p that targets dysferlin and protein kinase cAMP-activated catalytic subunit alpha

Abstract

Problem: To study the role of miRNA(s) during trophoblastic BeWo cell fusion.

Method of study: Changes in miRNA(s) profile of BeWo cells treated with forskolin were analyzed using Affymetrix miRNA microarray platform. Down-regulated miRNA, miR-92a-1-5p, was overexpressed in BeWo cells followed by forskolin treatment to understand its relevance in the process of BeWo cell fusion by desmoplakin I+II staining and hCG secretion by ELISA. Predicted targets of miR-92a-1-5p were also confirmed by qRT-PCR/Western blotting.

Results: The miRNA profiling of BeWo cells after forskolin (25 μmol/L) treatment identified miR-92a-1-5p as the most significantly down-regulated miRNA both at 24 and 48 hours time points. Overexpression of miR-92a-1-5p in these cells led to a significant decrease in forskolin-mediated cell fusion and hCG secretion. miRNA target prediction software, TargetScan, revealed dysferlin (DYSF) and protein kinase cAMP-activated catalytic subunit alpha (PRKACA), as target genes of miR-92a-1-5p. Overexpression of miR-92a-1-5p in BeWo cells showed reduction in forskolin-induced transcripts for DYSF and PRKACA. Further, reduction in DYSF (~2.6-fold) at protein level and PRKACA-encoded protein kinase A catalytic subunit alpha (PKAC-α; ~1.6-fold) were also observed.

Conclusion: These observations suggest that miR-92a-1-5p regulates forskolin-mediated BeWo cell fusion and hCG secretion by regulating PKA signaling pathway and dysferlin expression.

Keywords: dysferlin; forskolin-mediated BeWo cell fusion; miR-92a-1-5p; protein kinase A catalytic subunit alpha.