LINC00473 downregulation facilitates trophoblast cell migration and invasion via the miR-15a-5p/LITAF axis in pre-eclampsia.

More and more evidence has identified that long non-coding RNAs (lncRNAs) are involved in various biological process of numerous diseases. It has been reported that long intergenic non-protein coding RNA 473 (LINC00473) was associated with pre-eclampsia (PE) development. However, role and molecular mechanism of LINC00473 in PE remains elusive. Therefore, we designed this research to figure out the specific biological function of LINC00473 in trophoblasts. Firstly, we testified expressions of LINC00473 in trophoblasts of PE with RT-qPCR analysis. Then, to probe biological function of LINC00473 in trophoblasts of PE, CCK-8 assay, trans-well assays and western blot analysis were conducted in Wish and JAR cells. As for verifying interaction of microRNA-15a-5p (miR-15a-5p) and LINC00473 or lipopolysaccharide induced TNF factor (LITAF), RNA pull-down and luciferase reporter assays were carried out. Finally, rescue experiments were conducted to probe regulatory pattern of the LINC00473/miR-15a-5p/LITAF axis in trophoblasts of PE. As a result, LINC00473 presented a significant upregulation in trophoblasts of PE. Moreover, LINC00473 knockdown induced trophoblast viability, migration, invasion, and epithelial-to-mesenchymal transition (EMT) in trophoblasts. Additionally, miR-15a-5p interacted with LINC00473 and miR-15a-5p was negatively regulated by LINC00473 in trophoblasts. Simultaneously, miR-15a-5p negatively modulated LITAF in trophoblasts. Moreover, LITAF overexpression or miR-15a-5p downregulation reversed the promotive impact of silenced LINC00473 on trophoblast viability, migration, invasion and EMT. In conclusion, LINC00473 regulated migration and invasion in trophoblasts via the miR-15a-5p/LITAF axis. Our study may provide a novel insight for clinical treatment of PE.

Circ_0007121 Facilitates Trophoblastic Cell Proliferation, Migration, and Invasion via the Regulation of the miR-421/ZEB1 Axis in Preeclampsia.

Noncoding circular RNAs (circRNAs) have participated in the progression of preeclampsia (PE) via inhibiting microRNAs (miRNAs) to regulate gene expression. This study was designed to explore the miRNA/mRNA mechanism of hsa_circ_0007121 (circ_0007121) in PE. The expression detection of circ_0007121, microRNA-421 (miR-421), and zinc finger E-box binding homeobox 1 (ZEB1) was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. Transwell assay was used to determine cell migration and invasion. Cell apoptosis was evaluated using flow cytometry. The protein levels of epithelial-mesenchymal transition (EMT) markers and ZEB1 were measured via western blot. The interaction between miR-421 and circ_0007121 or ZEB1 was validated by dual-luciferase reporter assay. The expression detection indicated that circ_0007121 was downregulated in PE patients and the clinical data revealed that circ_0007121 was related to PE. The upregulation of circ_0007121 promoted cell proliferation, migration, invasion, and EMT in trophoblastic cells. Furthermore, circ_0007121 was identified as a sponge of miR-421 and the function of circ_0007121 was dependent on the sponge effect on miR-421. Moreover, ZEB1 was a target of miR-421 and circ_0007121/miR-421 axis could regulate the expression of ZEB1. In addition, miR-421 overexpression repressed trophoblastic cell behaviors through downregulating the ZEB1 level. Altogether, circ_0007121 contributed to the development of trophoblastic cells by regulating the miR-421/ZEB1 axis.

Pseudogene fms-related tyrosine kinase 1 pseudogene 1 (FLT1P1) cooperates with RNA binding protein dyskeratosis congenita 1 (DKC1) to restrain trophoblast cell proliferation and angiogenesis by targeting fms-related tyrosine kinase 1 (FLT1) in preeclampsia.

In preeclampsia (PE), preexistent maternal endothelial dysfunction leads to impaired placentation and vascular maladaptation. Long noncoding RNAs (lncRNAs) have been shown to participate in the placentation process. LncRNA fms-related tyrosine kinase 1 pseudogene 1 (FLT1P1) was previously reported to be upregulated in PE. In this study, we verified the effect of FLT1P1 and its cognate gene FLT1 on trophoblast cell proliferation and angiogenesis by using Cell Counting Kit-8 (CCK-8) assay, tube formation assay, and western blot analysis. Their binding to RNA binding protein dyskeratosis congenita 1 (DKC1) was validated by conducting RNA immunoprecipitation (RIP) and RNA pulldown assays. In this study, knockdown of FLT1P1 or FLT1 was found to promote cell proliferation and angiogenesis in trophoblasts. In addition, FLT1P1 recruited DKC1 to stabilize FLT1. Importantly, silencing FLT1P1 or DKC1 decreased the stability of FLT1. Rescue assays revealed that FLT1 overexpression reversed the effect of silenced FLT1P1. Overall, FLT1P1 cooperates with DKC1 to restrain trophoblast cell proliferation and angiogenesis by targeting FLT1.

microRNA-21 regulates the proliferation of placental cells via FOXM1 in preeclampsia.

The present study determined the expression of microRNA (miRNA or miR)-21 and forkhead box M1 (FOXM1) in placenta and blood samples from patients with preeclampsia (PE), and investigated the relationship between miR-21 and FOXM1. A total of 32 pregnant women with PE and 28 healthy pregnant women were included in the study as the experimental and control groups, respectively. Placental tissues and peripheral blood were collected from all subjects. ELISA was performed to measure the level of FOXM1 protein in the blood. HTR8/SVneo cells overexpressing miR-21 were established by transfection with agomiR-21. Reverse transcription-quantitative PCR was performed to measure the expression of FOXM1 mRNA and miR-21 in the placenta, blood and cells, and western blotting was used to evaluate FOXM1 protein expression in the placenta. An MTT assay was also performed to assess cell viability. In addition, a dual-luciferase reporter assay was used to investigate the direct interaction between FOXM1 and miR-21. The occurrence of PE was found to be associated with reduced FOXM1 mRNA levels, and elevated FOXM1 protein expression may serve a regulatory role that when attenuated leads to the occurrence of PE. Furthermore, miR-21 may serve a regulatory role in the pathology of PE by downregulating FOXM1 expression at the transcriptional level. In HTR8/SVneo cells, the overexpression of miR-21 reduced cell viability, possibly via the reduction of FOXM1 expression. The dual-luciferase assay indicated that miR-21 directly binds to the 3'-untranslated region of FOXM1 to regulate its expression. The present study demonstrated that the expression of FOXM1 mRNA and protein is downregulated, whereas the expression of miR-21 is upregulated in the placenta and blood samples of PE patients. In conclusion, miR-21 may regulate placental cell proliferation via its effects on FOXM1 to promote the occurrence and development of PE.

Long noncoding RNA SNHG12 promotes the proliferation, migration, and invasion of trophoblast cells by regulating the epithelial-mesenchymal transition and cell cycle.

OBJECTIVE: The deficient placental blood perfusion caused by the attenuated infiltration of trophoblast cells is a key factor in the occurrence of preeclampsia (PE). Furthermore, the long noncoding (lnc)RNA SNHG12 (small nucleolar RNA host gene 12) can promote the proliferation and metastasis of multiple tumor cells. However, whether lncRNA SNHG12 affects proliferation and metastasis of trophoblast cells is unclear. METHODS: We examined the level of lncRNA SNHG12 in plasma and placenta of patients with PE and constructed trophoblast cells with overexpressed or knocked down SNHG12. CCK-8, wound healing, and Transwell assays were used to detect alterations in proliferation, migration, and invasion of trophoblast cells. Western blotting was used to detect proteins related to the epithelial-mesenchymal transition (EMT), and cell cycle assays clarified cell cycle distribution. RESULTS: LncRNA SNHG12 promoted the proliferation, migration, and invasion of trophoblast cells. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, ß-catenin, and vimentin were positively correlated with SNHG12, and expression of E-cadherin was negatively correlated with SNHG12. SNHG12 also promoted the transition of trophoblast cells from G0/G1 to S phase. CONCLUSION: Overall, lncRNA SNHG12 promoted the migration and invasion of trophoblast cells by inducing the progression of EMT.

Mechanism of pomegranate ellagic polyphenols reducing insulin resistance on gestational diabetes mellitus rats.

BACKGROUND: Pomegranate ellagic polyphenols (PEP) has been used as a good medicine in many cultures throughout history. However, the mechanism of PEP regulated insulin resistance on gestational diabetes mellitus (GDM) rats is unclear. The main purpose of the present study was to explore the efficacy and mechanisms of PEP regulated in GDM rats. MATERIALS AND METHODS: Then, ELISA assay indicated that the levels of serum RBP4, Hcy, GA and FFA were lower in PEP groups than GDM groups in a dose-dependent manner. TUNEL staining showed that PEP improved the pathological changes and inhibited the cell apoptosis in the pancreatic and placenta tissues, respectively. RESULTS: We found that PEP improved the weight of pregnant rats and fetal rats and the level of blood glucose, blood biochemical index, insulin resistance in GDM rats. Results from H&E and immunohistochemical analysis found that PEP decreased the expressions of APN and Chemerin. Further, PEP decreased the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP), and increased the levels of 11ß-hydroxy steroid dehydrogenase type 2 (11ß-HSD2) and PPARα-TRB3-AKT2-p-FOXO1-GLUT2 signal related to insulin sensitivity in a dose-dependent manner. CONCLUSIONS: In conclusion, we have demonstrated that PEP may be a candidate drug for the treatment of GDM and guide the clinical therapy.

MicroRNA-298 inhibits malignant phenotypes of epithelial ovarian cancer by regulating the expression of EZH2.

MicroRNA (miRNA or miR)-298 has been reported to be downregulated and to modify the expression of the polycomb protein enhancer of zeste 2 (EZH2) in recurrent epithelial ovarian cancer (EOC). To date, no functional evidence of a miR-298-EZH2 axis in EOC has been documented. The present study aimed to investigate the associations of miR-298 and/or EZH2 expression with clinicopathological features of EOC patients, and revealed their roles in cell motility based on EOC cell lines. Reverse transcription-quantitative polymerase chain reaction was performed to detect the expression levels of miR-298 and EZH2 messenger RNA in human EOC tissues and cell lines. Wound healing and transwell assays were performed to determine the function of the miR-298-EZH2 axis on cell migration and invasion, respectively. Compared with normal tissues, miR-298 expression was significantly downregulated, while EZH2 expression was significantly upregulated, in human EOC tissues (both P=0.001). In addition, miR-298 downregulation and EZH2 upregulation were significantly associated with high clinical stage (both P=0.01) and pathological grade (both P=0.02) of EOC patients. Furthermore, the ectopic expression of miR-298 could efficiently inhibit cell migration and invasion. Notably, the overexpression of EZH2 could restore the cell migration and invasion abilities suppressed by miR-298. Our data offer convincing evidence that the dysregulation of the miR-298-EZH2 axis may be important in tumor progression of EOC patients. The present study also confirmed a tumor-suppressive role of miR-298 in modulating EOC cell motility by regulating the expression of EZH2, implying its potential as a novel miRNA-based therapeutic target for the treatment of human EOC.