Circular RNA VRK1 facilitates pre-eclampsia progression via sponging miR-221-3P to regulate PTEN/Akt.

Pre-eclampsia (PE) is a worldwide pregnancy-related disorder. It is mainly characterized by defect migration and invasion of trophoblast cells. Recently, circular RNAs (circRNAs) have been believed to play a vital role in PE. The expression patterns and the biological functions of circRNAs in PE remain elusive. Here, we performed a circRNA microarray to identify putative PE-related circRNAs. Bioinformatics analyses were used to screen the circRNAs which have potential relationships with pre-eclampsia, and we identified a novel circRNA (circVRK1) that was up-regulated in PE placenta tissues. By using HTR-8/SVneo cells, circVRK1 knockdown significantly enhanced cell migration and invasion abilities, as well as epithelial-mesenchymal transition (EMT). Mechanistically, we found that circVRK1 and PTEN could function as the ceRNAs to miR-221-3p. Overexpression of miR-221-3p promoted cell migration, invasion and EMT via regulating PTEN. The cotransfection of miR-221-3p inhibitor or PTEN reversed the effect from circVRK1 knockdown. Moreover, the circVRK1/miR-221-3p/PTEN axis greatly regulated Akt phosphorylation. In general, circVRK1 suppresses trophoblast cell migration, invasion and EMT, by acting as a ceRNA to miR-221-3p to regulate PTEN, and further inhibit PI3K/Akt activation. The purpose of this paper is to open wide insights to investigate the onset of PE and provide new potential therapeutic targets in PE.

Cyclin G2 upregulation impairs migration, invasion, and network formation through RNF123/Dvl2/JNK signaling in the trophoblast cell line HTR8/SVneo, a possible role in preeclampsia.

Disruption of extravillous trophoblast (EVT) migration and invasion is considered to be responsible for pathological placentation in preeclampsia (PE). Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression. However, its biological function and underlying molecular mechanism in PE are poorly understood. In this study, clinical data demonstrated that CCNG2 was significantly upregulated in PE placenta and associated with invasive EVT dysfunction. Additionally, Ccng2 knockout led to an attenuation of PE-like symptoms in the PE mouse model produced via treatment with NG-nitro-L-arginine methyl ester (L-NAME). In vitro, CCNG2 inhibited the migration, invasion, and endothelial-like network formation of human trophoblast cell line HTR8/SVneo. Mechanically, CCNG2 suppressed JNK-dependent Wnt/PCP signaling and its downstream indicators including epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) via promoting the polyubiquitination degradation of dishevelled 2 (Dvl2) protein in HTR8/SVneo cells. We also discovered that the E3 ligase Ring finger protein 123 (RNF123), as a novel CCNG2 target among HTR8/SVneo cells, interacted with Dvl2 and participated in CCNG2-induced polyubiquitination degradation of Dvl2. Moreover, we verified that the treatment of HTR8/SVneo cells with RNF123-specific siRNA improved polyubiquitination-induced degradation of Dvl2 and the activity of Wnt/PCP-JNK signaling mediated by CCNG2. Taken together, our results reveal that the CCNG2/RNF123/Dvl2/JNK axis may be involved in the pathogenesis and progression of PE through trophoblastic cell function modulation, thus probably providing us with new therapeutic strategies for PE treatment.

Association Between TLR4 Gene Polymorphisms and Risk of Preeclampsia: Systematic Review and Meta-Analysis.

BACKGROUND Toll-like receptor 4 (TLR4) plays a pivotal role in the innate immune response and is hyperactivated in preeclampsia (PE). Several researchers have published conflicting evidence for TLR4 rs4986790 and rs4986791 single nucleotide polymorphisms (SNPs) as risk factors for PE. The present meta-analysis was conducted to obtain a more definitive conclusion about the effects of these SNPs on PE susceptibility. MATERIAL AND METHODS To determine the correlation between rs4986790 and rs4986791 polymorphisms in the TLR4 gene and susceptibility to PE, the PubMed, Web of Science, EMBASE, Chinese National Knowledge Infrastructure, and Chinese WANFANG databases were searched for eligible articles. Statistical analysis was performed with STATA software, version 12.0. Pooled odds ratios with corresponding 95% confidence intervals (CIs) were extracted for assessment of correlation strength. RESULTS We identified 5 studies including 578 cases and 631 controls for the rs4986790 SNP and 4 studies including 469 cases and 457 controls for the rs4986791 SNP, mainly from a White population. The pooled analyses showed no statistical relationship between the polymorphisms rs4986790 and rs4986791 and PE susceptibility in 5 genetic models (all P>0.05). Moreover, the allelic and dominant gene models of rs4986790 and the allelic, heterozygous, and dominant gene models of rs4986791 had high heterogeneity. The sensitivity analysis explored potential sources of heterogeneity and confirmed the findings of this meta-analysis. CONCLUSIONS TLR4 rs4986790 and rs4986791 polymorphisms may not be implicated in PE susceptibility, primarily in a White population. More high-quality studies of genetic associations with PE are warranted.

Cyclin G2 regulates canonical Wnt signalling via interaction with Dapper1 to attenuate tubulointerstitial fibrosis in diabetic nephropathy.

Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. Cyclin G2 in the occurrence and development of diabetic nephropathy (DN), one of the most severe diabetic complications, has not been fully identified. In this study, we investigated the function and regulatory mechanism of cyclin G2 in DN. In vivo studies revealed that a deficiency of cyclin G2 significantly increased albuminuria and promoted tubulointerstitial fibrosis in established DN. Cyclin G2 regulated the expression of fibrosis-related proteins via the canonical Wnt signalling pathway in renal tubular epithelial cells. Moreover, the binding of cyclin G2 to Dapper1 (Dpr1/DACT1), a protein involved in Wnt signalling, decreased the phosphorylation of Dpr1 at Ser762 by casein kinase 1 (CK1) and suppressed the Wnt signalling pathway. These findings reveal that cyclin G2 can protect against renal injury and fibrosis associated with DN and, thus, is a new target for the prevention and treatment of diabetic complications.

Cyclin G2 Is Involved in the Proliferation of Placental Trophoblast Cells and Their Interactions with Endothelial Cells.

BACKGROUND Remodeling of maternal spiral arteries after embryo implantation relies on well-regulated trophoblast functions. Although cyclin G2 (CCNG2) is thought to be involved in placental development and function, its role in trophoblasts and the mechanisms underlying placental development and function remain unclear. The present study investigated the potential role of CCNG2 in trophoblast cell proliferation and their interactions with endothelial cells. MATERIAL AND METHODS CCNG2 levels were modified by stable infection of HTR8/SVneo cells with lentiviruses overexpressing and silencing CCNG2. Cell proliferation was measured using CCK-8 assays. Network formation assays were performed using trophoblasts alone and co-cultured trophoblasts and endothelial cells to measure angiogenesis of trophoblasts and trophoblast-endothelial interactions. Levels of angiogenic factors (VEGF and sFlt-1) in the supernatant were measured by ELISA, and the expression of cell cycle regulatory (cyclin D1) and invasive (MMP2, MMP3, MMP9) markers implicated in artery remodeling were measured by western blotting. RESULTS Ectopic expression of CCNG2 blocked the proliferation of HTR8/SVneo cells, as well as their abilities to form networks and integrate into human umbilical vein endothelial cells, whereas CCNG2 inhibition had the opposite effects. CCNG2 upregulation significantly reduced the expression of VEGF, cyclin D1, MMP2, MMP3, and MMP9, but enhanced the expression of sFlt-1. In contrast, CCNG2 downregulation had the opposite effects. CONCLUSIONS CCNG2 plays a critical role in trophoblast proliferation and trophoblast-endothelial cell interactions by significant affecting cell cycle, angiogenic, and invasive markers. CCNG2 may thus be a novel marker for the treatment of placental disorders.

MicroRNA-34a inhibits human trophoblast cell invasion by targeting MYC.

BACKGROUND: Preeclampsia, one of the major disorders of pregnancy, is characterized by inadequate trophoblast invasion and defective trophoblast-mediated remodeling of placental vasculature. MicroRNA-34a (miR-34a) has been found to be aberrantly expressed in the placentas of preeclamptic patients, yet its role in placental development and in the pathogenesis of preeclampsia remains elusive. RESULTS: The levels of miR-34a in the placentas of 20 preeclamptic patients and 20 healthy subjects were determined by real time-PCR, and miR-34a was found significantly elevated in the preeclamptic placentas. Further, the function of miR-34a in trophoblast cells was investigated by overexpressing miR-34a in JEG-3 trophoblast cell line. Overexpression of miR-34a in JEG-3 cells inhibited cell proliferation, migration and invasion. In addition, elevated expression of miR-34a reduced the expression of both endogenous and ectopic MYC. Moreover, we identified that MYC mRNA was a direct target of miR-34a in JEG-3 cells by dual luciferase reporter assay, and found that downregulation of MYC expression by miR-34a targeting significantly reduced the invasiveness of JEG-3 cells. CONCLUSIONS: Our findings provide preliminary evidence for the diverse functions of miR-34a in trophoblast biology, and suggest that miR-34a suppresses trophoblast invasion by directly targeting MYC.

Divergence of Iodine and Thyroid Hormones in the Fetal and Maternal Parts of Human-Term Placenta.

The human placenta is an important organ that forms a barrier where maternal and fetal exchange takes place. The placenta transport iodine to the fetal circulation by transfer of maternal iodine and deiodination of thyroid hormones (THs). The aim of the study was to examine the distribution of iodine and thyroid hormone transporters in the maternal and fetal sides of human-term placenta. A cross-sectional study was performed at the First Affiliated Hospital of China Medical University. Placental samples (maternal and fetal surfaces) were collected from 113 healthy-term pregnant women. The iodine content; the concentration of thyroxine (T4), triiodothyronine (T3), and reverse T3 (rT3); and the enzyme activity of placental type 2 iodothyronine deiodinase (D2) and D3 were examined. The mRNA and protein localization/expression of iodine and thyroid hormone transporters in the placenta were also studied. We also analyzed the association between expression level of Na+/I- symporter (NIS), thyroid hormone transporter protein, D3 activity in maternal and fetal surfaces of placenta with iodine content, and thyroid hormone levels. Iodine levels in placental samples from the maternal side were significantly higher than those in samples from the fetal side. T3 and T4 expression in fetal placenta was significantly lower than in maternal placenta. D3 activity in the fetal side of the placentas was significantly higher than that in the maternal side. The mRNA and protein expression of monocarboxylate transporters 8 (MCT8), L-amino acid transporters 1 (LAT1), organic anion transporting polypeptides 4A1 (OATP4A1), and TH binding protein transthyretin (TTR) were significantly increased in maternal side, while the NIS expression was higher in fetal side of human-term placenta. In conclusion, the enzymatic deiodination of thyroid hormones forms a barrier which reduces transplacental passage of the hormones and that the maternal part of the placenta is the primary factor in the mechanism regulating the hormonal transfer.

Corrigendum to "Cyclin G2 Suppresses Glomerulosclerosis by Regulating Canonical Wnt Signalling".

[This corrects the article DOI: 10.1155/2018/6938482.].

Plasma levels of TAM receptors and ligands in severe preeclampsia.

BACKGROUND: Preeclampsia (PE) is a multifactorial dysfunction characterized by hypertension with characteristics of systematic endothelial activation. It is widely accepted that vascular disorder and deficient trophoblast invasion are involved in PE. Tyro3/Axl/MerTK (TAM) family receptors are tyrosine-kinase receptors and may exert a diverse range of functions such as cell proliferation, migration, and vascular angiogenesis. The role of TAM signaling in severe PE patients remains unclear. Therefore, the aim of this study was to investigate involvement of the TAM axis in the pathogenesis of PE. METHODS: A total of 36 severe PE patients and 40 age- and gender-matched healthy pregnant women (controls) were enrolled in this study. Plasma levels of soluble TAM receptors (Tyro3, Axl, MerTK) and ligands (Gas6 and ProS) were then measured using an enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the expression of these proteins and the clinical features of PE. RESULTS: Plasma levels of sMerTK and sAxl were significantly higher in severe PE patients than in control women during pregnancy. The plasma concentrations of sMerTK and sAxl in severe PE patients correlated positively with systolic and diastolic blood pressure, and plasma sAxl levels demonstrated a significant correlation to proteinuria. In contrast, reduced levels of Gas6 were inversely associated with urine protein in PE patients. CONCLUSIONS: Elevated expression of the plasma levels of sMerTK and sAxl, as well as the reduction of Gas6 were observed in severe PE patients. Furthermore, these changes were correlated with disease activity. TAM signaling might play a role in the pathogenesis of PE.

Cyclin G2 Suppresses Glomerulosclerosis by Regulating Canonical Wnt Signalling.

Recent data has shown that cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. The involvement of cyclin G2 in the occurrence and development of diabetic nephropathy (DN) has not been determined. In the present study, we conducted cyclin G2 knockout studies to determine whether this protein regulates glomerulosclerosis in DN mice. We found that cyclin G2 regulated the expression of renal glomerulosclerosis-related proteins via the canonical Wnt signalling pathway in glomerular mesangial cells. A cyclin G2 deficiency resulted in more severe renal injury in DN mice. These findings provided new insight into the pathogenesis of DN, revealing that cyclin G2 has a protective role in glomerulosclerosis and is a potential new target for the prevention and treatment of DN.

Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion.

Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1.

Long non-coding RNA MALAT-1 is downregulated in preeclampsia and regulates proliferation, apoptosis, migration and invasion of JEG-3 trophoblast cells.

Long non-coding RNA (lncRNA), as a newly identified subset of the transcriptome, has been implicated in a variety of physiological and pathological processes. Metastasis associated lung adenocarcinoma transcript-1 (MALAT-1), a lncRNA that was initially detected in the metastatic lung cancer, was reported to be overexpressed in placenta previa increta/percreta (I/P), which is caused by excessive trophoblast invasion. However, the role of MALAT-1 in the regulation of trophoblast behavior is not fully understood. In this study, we first examined the expression of MALAT-1 in the placentas from the patients with preeclampsia, the pathology of which is associated with inadequate trophoblast invasion, and found that the expression of MALAT-1 was downregulated in the preeclamptic placentas as compared to the normal placentas. We further investigated the function of MALAT-1 in JEG-3 trophoblast cell line using short interfering RNA (siRNA) against MALAT-1 transcripts. Silencing of MALAT-1 in JEG-3 cells suppressed proliferation and induced cell cycle arrest at G0/G1 phase. Reduced expression of MALAT-1 by RNA interference resulted in enhanced apoptosis in JEG-3 cells, accompanied with elevated levels of the pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). Moreover, the migration rate and the invasiveness of JEG-3 cells were suppressed when MALAT-1 was downregulated. In summary, our results suggest that MALAT-1 may play an important role in the regulation of proliferation, cell cycle, apoptosis, migration and invasion of trophoblast cells, and under-expression of MALAT-1 during early placentation may be involved in the pathogenesis of preeclampsia.

Endothelial nitric oxide synthase gene polymorphisms (G894T, 4b/a and T-786C) and preeclampsia: meta-analysis of 18 case-control studies.

Studies investigating the association between endothelial nitric oxide synthase (eNOS) gene polymorphisms and preeclampsia reported contradictory or nonconclusive results. We performed a meta-analysis of 18 genetic association studies that examined the relationship between preeclampsia and the G894T, 4a/b and T-786C polymorphisms of the eNOS gene. Subgroup analysis by ethnicity and potential sources of heterogeneity and bias were explored. The MEDLINE, EMBASE, and Google Scholar databases were searched to access the relevant genetic association studies up to June 2011. For the allelic analysis of the G894T variant, all studies showed no significant association. For the genotypic analysis, the combined studies of the G allele showed negative significance (odds ratio [OR]=0.56; 95% confidence interval [CI]: 0.33-0.97), all the studies showed positively significance when the T allele was combined (OR=1.17; 95% CI: 1.01-1.36), and results were also positively significant in non-Asian populations (OR=1.20; 95% CI: 1.02-1.43). For the allelic analysis of the 4b/a variant, all studies showed no significant association, but results were negatively significant in non-Asian populations (OR=0.67; 95% CI: 0.46-0.98). For the genotype analysis, combined studies of the b allele showed negative significance (OR=0.55; 95% CI: 0.36-0.84). Moreover, non-Asian studies showed negatively significant results (OR=0.45; 95% CI: 0.28-0.72). For the analysis of the T-786C variant, none of the studies showed significant results. The synthesis of available evidence supports the fact that intron 4a allele, homozygosity for the 894T and intron 4a of eNOS are positively associated with preeclampsia. Large, multiethnic confirmatory, and well-designed studies are needed to determine the relation between preeclampsia and polymorphisms of the eNOS gene.

Elevated expression of PEG10 in human placentas from preeclamptic pregnancies.

The aim of this study was to determine qualitative and quantitative changes in paternally expressed gene 10 (PEG10) expression in preeclamptic placentas. Placental tissues were obtained immediately after delivery from women with normal pregnancies (n=20) and patients with preeclampsia (n=20). Quantitative real-time RT-PCR, Western blot analysis and immunohistochemistry were used to determine PEG10 gene expression and localization in placental tissues. Compared with the normal group, PEG10 was highly expressed at both mRNA and protein levels in preeclampsia (P<0.05). In immunohistochemical staining, PEG10 was present in the syncytiotrophoblast, cytotrophoblast, endothelial cell and stroma of all placentas. Notably, the intensity of PEG10 expression in the syncytiotrophoblast, cytotrophoblast and stem villi was much higher in preeclampsia than in normal. In conclusion, elevated expression of PEG10 is likely to be involved in the pathophysiology of preeclampsia. Further studies are needed to elucidate the precise role of PEG10 in preeclampsia.

Identification of differential gene expression profiles in placentas from preeclamptic pregnancies versus normal pregnancies by DNA microarrays.

The purpose of this study was to perform a comprehensive analysis of gene expression profiles in placentas from preeclamptic pregnancies versus normal placentas. Placental tissues were obtained immediately after delivery from women with normal pregnancies (n=6) and patients with preeclampsia (n=6). The gene expression profile was assessed by oligonucleotide-based DNA microarrays and validated by quantitative real-time RT-PCR. Functional relationships and canonical pathways/networks of differentially-expressed genes were evaluated by GeneSpring™ GX 11.0 software, and ingenuity pathways analysis (IPA). A total of 939 genes were identified that differed significantly in expression: 483 genes were upregulated and 456 genes were downregulated in preeclamptic placentas compared with normal placentas (fold change ≥ 2 and p<0.05 by unpaired t-test corrected with Bonferroni multiple testing). The IPA revealed that the primary molecular functions of these genes are involved in cellular function and maintenance, cellular development, cell signaling, and lipid metabolism. Pathway analysis provided evidence that a number of biological pathways, including Notch, Wnt, NF-κB, and transforming growth factor-ß (TGF-ß) signaling pathways, were aberrantly regulated in preeclampsia. In conclusion, our microarray analysis represents a comprehensive list of placental gene expression profiles and various dysregulated signaling pathways that are altered in preeclampsia. These observations may provide the basis for developing novel predictive, diagnostic, and prognostic biomarkers of preeclampsia to improve reproductive outcomes and reduce the risk for subsequent cardiovascular disease.