Upregulation of circRNA hsa_circ_0008726 in Pre-eclampsia Inhibits Trophoblast Migration, Invasion, and EMT by Regulating miR-345-3p/RYBP Axis.Accumulating evidence shows that impaired spiral artery remodeling, placental dysfunction, and insufficient trophoblast infiltration contribute to the etiology and pathogenesis of pre-eclampsia (PE).  circRNAs are a class of endogenous non-coding RNAs implicated in the pathogenesis of many diseases, including PE.  This study aims to investigate the role of circRNA hsa_circ_0008726 in regulating the migration and invasion of extravillous trophoblast cells.  RNase R assay was performed to confirm that circ_0008726 was a circular transcript.  The expression of circ_0008726, RYBP, and miR-345-3p was examined by qRT-PCR.  The functional interaction between miR-345-3p and circ_0008726 or RYBP was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP).  Cell migration and invasion ability was analyzed by Transwell assays.  Western blot was used for the quantification of RYBP protein level.  Circ_0008726 expression was significantly increased in PE placenta tissues as compared with normal placenta tissues.  Circ_0008726 was resistant to RNase R digestion and was predominately located in the cytoplasm of HTR-8/SVneo cells.  Silencing circ_0008726 promoted cell migration and EMT (epithelial-mesenchymal transition), while circ_0008726 overexpression suppressed these processes.  Mechanistically, circ_0008726 sponged miR-345-3p to negatively regulate its expression, and miR-345-3p negatively modulated the expression of RYBP.  In PE samples, the expression level of circ_0008726 was negatively correlated with miR-345-3p level, but was positively correlated with RYBP expression.  Transfection of miR-345-3p mimic or RYBP knockdown counteracted the effects of circ_0008726 overexpression on cell migration and EMT.  Our data demonstrate the upregulation of circ_0008726 in PE placenta, which inhibits the migration, invasion, and EMT of HTR-8/SVneo cells by targeting miR-345-3p/RYBP axis.  These data suggest that circ_0008726 could be a potential biomarker and therapeutic target for PE.
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RNase R assay was performed to confirm that circ_0008726 was a circular transcript. 
RNase R assay
The expression of circ_0008726, RYBP, and miR-345-3p was examined by qRT-PCR. 
qRT-PCR
The functional interaction between miR-345-3p and circ_0008726 or RYBP was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP). 
Dual-luciferase reporter assay
The functional interaction between miR-345-3p and circ_0008726 or RYBP was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP). 
RNA immunoprecipitation (RIP)
Cell migration and invasion ability was analyzed by Transwell assays. 
Transwell assays
Western blot was used for the quantification of RYBP protein level. 
Western blot