Decreased circUBAP2 Expression Is Associated with Preeclampsia by Limiting Trophoblast Cell Proliferation and Migration

Abstract

Preeclampsia (PE) is a common obstetric disease and a major cause of maternal, newborn, and fetal death. This condition is a multisystem disorder characterized by hypertension, proteinuria, and involvement of the kidney, liver, and nervous system. It is generally believed that the placenta is the main cause of PE. circRNAs are a special class of noncoding RNAs that can form covalently closed continuous ring structures with tissue-specific conservation, and they have been reported to play a wide range of regulatory functions in various diseases, including PE. In this study, we reported a novel circUBAP2 (hsa_circ_0003496) and found that it was downregulated in placental tissues from patients with PE compared to healthy controls. After knocking down circUBAP2 in trophoblast cells, we found that cell proliferation and migration were significantly suppressed. In addition, preliminary mechanistic studies showed that circUBAP2 can sponge miR-1244, and FOXM1 was identified as a target gene for miR-1244. Cotransfection of si-circUBAP2 and a miR-1244 inhibitor partially reversed the suppressive effect induced by circUBAP2 depletion on proliferation and migration. In conclusion, the circUBAP2/miR-1244/FOXM1 axis might be a promising molecular marker for the diagnosis and treatment of PE.

Keywords: FOXM1; Placenta; Preeclampsia; circUBAP2; miR-1244.

Fig. 1

circRNA circUBAP2 was downregulated in the placental tissues of patients with PE. a Genomic scheme of circUBAP2. b Divergent primers were used to detect circular RNAs in cDNA but not gDNA, and GAPDH was used as a control. c Sanger sequence of the junction site of circUBAP2. d qPCR analysis of GAPDH and circUBAP2 after RNase R treatment. e The expression of circUBAP2 in placental samples of patients with PE and healthy controls was analyzed by qPCR. f The relative expression of circUBAP2 in the trophoblast cell line HTR-8/SVneo and trophoblastic tumor cell line JEG-3 was measured by qPCR. Data are expressed as the mean ± SD, unpaired t test, **p < 0.01

Fig. 2

Inhibition of circUBAP2 suppresses trophoblast cell proliferation. a The interference efficiency was examined by qPCR in HTR-8/SVneo and JEG-3 cells after transfection with siRNA (si-circ) targeting circUBAP2. b The viability of HTR-8/SVneo and JEG-3 cells was measured by CCK-8 assays after transfection with si-circ and si-NC for 24, 48, and 72 h. c, d Colony formation of HTR-8/SVneo (c) and JEG-3 (d) cells transfected with siRNAs. e, f EdU assay to determine the cell proliferation rate of HTR-8/SVneo (e) and JEG-3 (f) cells transfected with siRNAs. g, h Cell apoptosis rates were detected using flow cytometry. Data are expressed as the mean ± s.d., unpaired t-test, **p < 0.01

Fig. 3

Inhibition of circUBAP2 suppresses trophoblast cell migration. a, c Cell motility was examined in cells transfected with si-circ or si-NC by wound healing assays. b, d Transwell migration assays were performed in cells transfected with si-circ or si-NC. Data are expressed as the mean ± SD, unpaired t test, **p < 0.01

Fig. 4

circUBAP2 directly binds to miR-1244 to regulate FOXM1 expression. a Potential binding sites of miR-1244 on circUBAP2. b, c Luciferase reporter assay of wild-type/mutant circUBAP2 with miR-1244/miR-NC in HTR-8/SVneo (b) and JEG-3 (c) cells. d The relative expression of miR-1244 in trophoblast cells after transfection with si-circ or si-NC. e The predicted binding sites between FOXM1 and miR-1244. f The relative luciferase reporter activity of miR-1244 mimics/NC cotransfected with pmirGLO-WT/Mut-circUBAP2 in HTR-8/SVneo cells. g The relative expression of FOXM1 mRNA in trophoblast cells after transfection with miR-1244 mimic/NC or inhibitor/NC was measured by qPCR. h, i The relative expression of FOXM1 mRNA (h) and protein (i) in trophoblast cells after transfection with si-circ alone or cotransfection with miR-1244 inhibitor was measured by qPCR or western blots, respectively. Data are expressed as the mean ± SD, unpaired t test, *, compared with the si-NC group; #, compared with the si-circ group. *, #, p < 0.05, **, ##, p < 0.01

Fig. 5

circUBAP2 contributes to cell proliferation and migration by regulating miR-1244/FOXM1. a, b CCK-8 assay was performed to measure cell viability after transfection with si-NC, si-circ, or si-circ+inhibitor miR-1244 in HTR-8/SVneo (a) or JEG-3 (b) cells. c, d Cell migration was assessed by Transwell assays. Data are expressed as the mean ± SD, unpaired t test, *, compared with the si-NC group; #, compared with the si-circ group. #, p < 0.05, **, ##, p < 0.01