| BackgroundSpontaneous abortion(SA) is a common medical problem. Approximately 25% of women of reproductive age who become pregnant will experience SA. Early spontaneous abortion(ESA) occurs prior to 20 gestational weeks and is one of the most common reproductive failures of human pregnancy. The potential pathogenesis of SA includes foetal chromosomal abnormalities, endocrine disorders, and immune factors. However, how these events lead to ESA and the molecular mechanisms of ESA are still poorly understood.Trophoblasts are placental cells of epithelial lineage and terminally differentiate from common cytotrophoblast progenitors into highly specialized cells that are essential for placental development. As is well-known, several foetal and maternal disorders affect the placental development and function of trophoblasts, leading to severe pregnancy disorders, including ESA and pre-eclampsia.LncRNAs are conventionally defined transcript longer than 200 nucleotides that do not encode proteins, which were initially considered a byproduct of RNA polymerase II transcription and described as “dark matter” without biological function. However, whole-genome transcriptomic analyses have identified large numbers of dynamically expressed lncRNAs, many of which are involved in a variety of biological functions. lncRNAs have been increasingly shown to have functional roles in body development and tumourigenesis via the regulation of related gene expression.Furthmore, Most recently, the genome-wide differential expression of long noncoding RNAs(lncRNAs) was determined in the embryonic sac and decidua from spontaneous abortion(SA) and induced abortion(IA), reminded that lncRNA may play important roles in SA.And that lncRNA-H19 expression is increased in fetal liver and placenta. One study reported that one of the main physiological role of lncRNA-H19 is to limit growth of the placenta prior to birth via the regulation of mi R-675 processing. In addition, knockdown of the imprinting H19 gene or inhibition of miR-675 produced similar proliferation-promoting effects in human trophoblastic JEG-3 cells. However, the expression of miR-675 began to increase at the late stage of placental development, whereas the expression of H19 remained elevated at the early stage of placental development. Thus, two questions arise. What is the physiological and pathological significance of increased H19 expression in early placental development? What is the mechanism of action of H19 when the expression of mi R-675 is lost at the early stage of placental development?In this study, we analysed the expression of lncRNA-H19 in early SA and induced abortion(IA) samples. In addition,further verify the clinic significance of up-expression of H19. Finally, overexpression of H19 at the cellular level to study its effect on the regulation of human trophoblast cells and further explore the mechanism of action of H19.MethodsFirstly,we collected Villous tissues from patients with ESA and women undergoing selective pregnancy termination in the first trimester(7-9 weeks) for nonmedical reasons in the Changning district central hospital of Shanghai.Then we used of RNA isolation,inverse transcription and real-time PCR technology to test the expression of lncRNA-H19 in the villi tissue. In addition,we overexpression H19 in human trophoblast and using CCK-8 technology to detect the level of cell proliferation.And then use Transwell method(Millipore),Matrix Reagent(BD) to examine the change of H19 invasive capacility in human trophoblast cells.In order to further explore the mechanism, this research used RNA pull down technology and Mass Spectrometry filtering lncRNA-H19 related binding proteins in human trophoblast cells,and using Western blot,RIP technology and immunofluorescence technology to verify the relationship between H19 and related binding proteins.and then detected mechanism of H19. At the same time, the level of global DNA methylation in human trophoblast cells and the methylation level changes of H19 promoter region was determined respectively using the Methylation Quantification Ultra Kit(Epigentek, Farmingdale, NY, USA) and pyrophosphate seguencing technology.Results1.lncRNA-H19 was upregulated in the SA samples, consistent with the hypomethylation of the differential methylation region(DMR) of lncRNA-H19.The results showed that lncRNA-H19 was highly expressed in human villous tissue from early spontaneous abortion patients and(to a lesser extent) in human villous tissue from induced-abortion patients.The results showed that global DNA methylation was significantly reduced in villous tissue from women with early spontaneous abortion compared with the control group(women with induced abortion,). The results also showed that the promoter region of the lncRNA-H19 gene in villous tissue from women with early spontaneous abortion had lower DNA methylation levels compared with the control group.2.Overexpression of lncRNA-H19 inhibited trophoblast cell growth and invasion.Overexpression of lncRNA-H19 in HTR8/Svneo trophoblasts was identified, The results showed overexpression of H19 in HTR8/Svneo trophoblasts with increasing expression compared with the control group. The results showed that upregulation of lncRNA-H19 inhibited cell proliferation in HTR8/SVneo trophoblasts. Overexpression of lncRNA-H19 also showed decreased cell motility in HTR8/SVneo trophoblasts.3.To explore the molecular mechanism of lncRNA-H19 in early placental development, the RNA-binding proteins for lncRNA-H19 were screened by RNA pulldown and mass spectrometry. The protein DDX3 X was identified as the RNA-binding protein for lncRNA-H19.In the RNA pulldown assays that were performed to identify proteins associated with lncRNA-H19 RNA in HTR8/SVneo trophoblasts, DDX21 and DDX3 X were the main proteins identified by mass spectrometry, but in three independent RNA pulldown assays, only DDX3 X was detected by western blotting. The results also showed that lncRNA-H19 expression affected DDX3 X protein levels but not mRNA levels in HTR8/SVneo cells.4.Furthermore, our studies demonstrated that H19 inhibited Wnt-β-catenin pathway activation in trophoblasts via effects on DDX3 X function,which finally explain the mechanism of action of H19.The results showed overexpression of DDX3 X and decreased protein expression of E-cadherin. The results also suggested that the overexpression of DDX3 X in HTR8/SVneo cells promoted the translocation of β-catenin from the cytomembrane to the cell nucleus, which confirmed the effect of the activation of DDX3 X on the Wnt-β-catenin pathway. Furthermore, upregulation of lncRNA-H19 counteracted the impact of DDX3 X on the Wnt-β-catenin pathway.Conclusion1. Upregulated expression of lncRNA-H19 in the human villi of ESA;2. Promoter hypomethylation of lncRNA-H19 and genome-wide reduced DNA methylation in villi of ESA;3. Overexpression of lncRNA-H19 inhibits trophoblast proliferation and invasion;4. Identification of DDX3 X as the RNA-binding protein of lncRNA-H19 in trophoblast HTR-8/SVneo cells;5. LncRNA-H19 affects the function of DDX3 X in the activation of the Wnt-β-catenin pathway in human trophoblasts;... |
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