Circ-CCNB1 Modulates Trophoblast Proliferation and Invasion in Spontaneous Abortion by Regulating miR-223/SIAH1 axis.

CONTEXT: Spontaneous abortion (SA) is a common disorder in early pregnancy. Circular RNAs (circRNAs) have been reported to exert important regulatory effects on trophoblast function and embryo development. OBJECTIVE: The aim of this study was to explore whether and how circRNAs regulate trophoblast function in SA during early pregnancy. METHODS: Cell proliferation, 5-bromo-2-deoxyuridine (BrdU) staining, Transwell, immunofluorescence, Western blot, RNA pull-down, and dual luciferase reporter assays were performed to investigate the effect of circRNA cyclin B1 (circ-CCNB1) on trophoblast function in HTR-8/SVneo and JEG-3 cells. RESULTS: An in vitro study demonstrated that upregulation of circ-CCNB1 significantly inhibited trophoblast proliferation and invasion compared with the controls using HTR-8/SVneo and JEG-3 cells, respectively. Moreover, miR-223 was downregulated in the villous tissues of patients with SA and was further predicted and shown to negatively interact with circ-CCNB1, which is involved in trophoblast proliferation and invasion. Using bioinformatics tools and subsequent RNA pull-down and dual luciferase assays, we found that miR-223 directly targets seven in absentia homolog-1 (SIAH1) and that upregulation of miR-223 decreased circ-CCNB1-induced SIAH1 expression levels in HTR-8/SVneo cells. Interestingly, upregulation of circ-CCNB1 suppressed trophoblast proliferation and invasion through inhibition of CCNB1 nuclear translocation induced by SIAH1. Downregulation of SIAH1 enhanced circ-CCNB1-suppressed CCNB1 nuclear protein expression in trophoblast cells. CONCLUSION: Circ-CCNB1 served as a modulator of trophoblast proliferation and invasion by sponging miR-223, thus forming a regulatory network of circ-CCNB1/miR-223/SIAH1 in modulating CCNB1 nuclear translocation, which enabled us to elucidate the molecular mechanisms involved in normal embryo implantation or in SA.

[Analysis of primary metabolites of ranolazine in dog urine by LC-MS(n)].

Ranolazine and metabolites in dog urine were identified by LC-MS(n). Dog urine samples were collected after ig 30 mg x kg(-1) ranolazine, then the samples were enriched and purified through solid-phase extraction cartridge. The purified samples were analyzed by LC-MS(n). The possible metabolites were discovered by comparing the full scan and SIM chromatograms of the test samples with the corresponding blanks. Seventeen phase I metabolites and fourteen phase II metabolites were identified in dog urine. Three metabolites were identified by comparing with the control article. The metabolites were formed via the following metabolic pathways: O-demethylation, O-dearylation, hydroxylation, N-dealkylation, amide hydrolysis, glucuronidation and sulfation. The LC-MS(n) method is suitable for the rapid identification of drug and its metabolites in biologic samples.