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• W3089169813 abstract "The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE. The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE. Preeclampsia (PE) is a multisystem disorder that affects about 2%–7% pregnancies worldwide and is considered one of the leading causes of maternal and neonatal mortality as well as morbidity.1Steegers E.A. von Dadelszen P. Duvekot J.J. Pijnenborg R. Pre-eclampsia.Lancet. 2010; 376: 631-644Abstract Full Text Full Text PDF PubMed Scopus (2116) Google Scholar,2Mol B.W.J. Roberts C.T. Thangaratinam S. Magee L.A. de Groot C.J.M. Hofmeyr G.J. Pre-eclampsia.Lancet. 2016; 387: 999-1011Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar Although the etiology and the pathogenesis of PE are not well understood, it is generally accepted that placental defects, especially the dysregulation of the trophoblast behaviors, are the underlying mechanisms that lead to the disease. For instance, reduced trophoblast proliferation, excessive trophoblast cell apoptosis, as well as the insufficient trophoblast invasion have all been linked to the occurrence and the development of PE.3Lyall F. Robson S.C. Bulmer J.N. Spiral artery remodeling and trophoblast invasion in preeclampsia and fetal growth restriction: relationship to clinical outcome.Hypertension. 2013; 62: 1046-1054Crossref PubMed Scopus (265) Google Scholar,4Chen J.Z. Sheehan P.M. Brennecke S.P. Keogh R.J. Vessel remodelling, pregnancy hormones and extravillous trophoblast function.Mol. Cell. Endocrinol. 2012; 349: 138-144Crossref PubMed Scopus (84) Google Scholar MicroRNAs (miRNAs) are a subset of small, noncoding RNAs that are involved in numerous important biological events.5Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (28615) Google Scholar A major mechanism by which miRNAs regulate gene expression is the partial base pairing to the 3′ untranslated region (3′ UTR) of the target mRNAs to repress their translation and/or to induce their degradation.6Chekulaeva M. Filipowicz W. Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.Curr. Opin. Cell Biol. 2009; 21: 452-460Crossref PubMed Scopus (551) Google Scholar Our7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar and others’8Pineles B.L. Romero R. Montenegro D. Tarca A.L. Han Y.M. Kim Y.M. Draghici S. Espinoza J. Kusanovic J.P. Mittal P. et al.Distinct subsets of microRNAs are expressed differentially in the human placentas of patients with preeclampsia.Am. J. Obstet. Gynecol. 2007; 196: 261.e1-261.e6Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar,9Zhu X.M. Han T. Sargent I.L. Yin G.W. Yao Y.Q. Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies.Am. J. Obstet. Gynecol. 2009; 200: 661.e1-661.e7Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar previous work have demonstrated that a great number of miRNAs are differentially expressed in PE placentas, and some of these differentially expressed miRNAs may participate in the regulation of various trophoblast cell functions.7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar,10Pan Q. Niu H. Cheng L. Li X. Zhang Q. Ning Y. Invasion of trophoblast cell lines is inhibited by miR-93 via MMP-2.Placenta. 2017; 53: 48-53Crossref PubMed Scopus (15) Google Scholar,11Gao W.L. Liu M. Yang Y. Yang H. Liao Q. Bai Y. Li Y.X. Li D. Peng C. Wang Y.L. The imprinted H19 gene regulates human placental trophoblast cell proliferation via encoding miR-675 that targets Nodal Modulator 1 (NOMO1).RNA Biol. 2012; 9: 1002-1010Crossref PubMed Scopus (118) Google Scholar miR-18a is predominantly downregulated in placental tissues and maternal plasma of patients with PE and is mainly localized in the various subtypes of trophoblast cells in the placenta.7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar A series of in vitro and in vivo studies have demonstrated that miR-18a plays an important role in different cell functions via downregulating FGF1, Smad4, HIF-1α, and other target genes.12Liu C. Chen M. Wang M. Pi W. Li N. Meng Q. MiR-18a regulates myoblasts proliferation by targeting Fgf1.PLoS ONE. 2018; 13: e0201551PubMed Google Scholar, 13Montoya M.M. Maul J. Singh P.B. Pua H.H. Dahlström F. Wu N. Huang X. Ansel K.M. Baumjohann D. A Distinct Inhibitory Function for miR-18a in Th17 Cell Differentiation.J. Immunol. 2017; 199: 559-569Crossref PubMed Scopus (29) Google Scholar, 14Chen X. Wu L. Li D. Xu Y. Zhang L. Niu K. Kong R. Gu J. Xu Z. Chen Z. Sun J. Radiosensitizing effects of miR-18a-5p on lung cancer stem-like cells via downregulating both ATM and HIF-1α.Cancer Med. 2018; 7: 3834-3847Crossref PubMed Scopus (42) Google Scholar Therefore, elucidation of the underlying mechanisms by which miR-18a regulates the functions of trophoblast cells may provide a better understanding of the pathophysiology of this disorder and uncover new targets for therapeutic intervention. With the use of bioinformatics tool Targetscan,15Lewis B.P. Burge C.B. Bartel D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9644) Google Scholar,16Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4127) Google Scholar we found that Smad2 and Smad3 were both among the most predicted targets of miR-18a. Smad2 and Smad3 are both central cytoplasmic mediators that can be activated by transforming growth factor-β (TGF-β) and its specific serine/threonine kinase receptors.17Heldin C.H. Miyazono K. ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar,18Massagué J. TGFβ signalling in context.Nat. Rev. Mol. Cell Biol. 2012; 13: 616-630Crossref PubMed Scopus (2040) Google Scholar TGF-β is a pleiotropic factor that plays essential roles in regulating numerous physiological and pathological processes.19Goumans M.J. Liu Z. ten Dijke P. TGF-beta signaling in vascular biology and dysfunction.Cell Res. 2009; 19: 116-127Crossref PubMed Scopus (382) Google Scholar Furthermore, it is highly expressed in PE placentas,20Benian A. Madazli R. Aksu F. Uzun H. Aydin S. Plasma and placental levels of interleukin-10, transforming growth factor-beta1, and epithelial-cadherin in preeclampsia.Obstet. Gynecol. 2002; 100: 327-331Crossref PubMed Scopus (76) Google Scholar,21Caniggia I. Grisaru-Gravnosky S. Kuliszewsky M. Post M. Lye S.J. Inhibition of TGF-beta 3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies.J. Clin. Invest. 1999; 103: 1641-1650Crossref PubMed Scopus (301) Google Scholar and the dysregulation of its signaling pathway is responsible for the dysfunction of trophoblast cells and the pathophysiology of PE.21Caniggia I. Grisaru-Gravnosky S. Kuliszewsky M. Post M. Lye S.J. Inhibition of TGF-beta 3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies.J. Clin. Invest. 1999; 103: 1641-1650Crossref PubMed Scopus (301) Google Scholar, 22Karmakar S. Das C. Regulation of trophoblast invasion by IL-1beta and TGF-beta1.Am. J. Reprod. Immunol. 2002; 48: 210-219Crossref PubMed Scopus (109) Google Scholar, 23Cheng J.C. Chang H.M. Leung P.C.K. TGF-β1 Inhibits Human Trophoblast Cell Invasion by Upregulating Connective Tissue Growth Factor Expression.Endocrinology. 2017; 158: 3620-3628Crossref PubMed Scopus (24) Google Scholar, 24Zuo Y. Fu Z. Hu Y. Li Y. Xu Q. Sun D. Tan Y. Effects of transforming growth factor-β1 on the proliferation and invasion of the HTR-8/SVneo cell line.Oncol. Lett. 2014; 8: 2187-2192Crossref PubMed Scopus (11) Google Scholar, 25Xu J. Sivasubramaniyam T. Yinon Y. Tagliaferro A. Ray J. Nevo O. Post M. Caniggia I. Aberrant TGFβ Signaling Contributes to Altered Trophoblast Differentiation in Preeclampsia.Endocrinology. 2016; 157: 883-899Crossref PubMed Scopus (30) Google Scholar, 26Cheng J.C. Chang H.M. Leung P.C. Transforming growth factor-β1 inhibits trophoblast cell invasion by inducing Snail-mediated down-regulation of vascular endothelial-cadherin protein.J. Biol. Chem. 2013; 288: 33181-33192Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 27Zhao M.R. Qiu W. Li Y.X. Zhang Z.B. Li D. Wang Y.L. Dual effect of transforming growth factor beta1 on cell adhesion and invasion in human placenta trophoblast cells.Reproduction. 2006; 132: 333-341Crossref PubMed Scopus (51) Google Scholar We therefore speculated that miR-18a interferes with the TGF-β signaling in PE placentas via targeting Smad2 and/or Smad3. Smad2 is composed of 12 exons, and exon3 is spliced out in about 10% of Smad2 in several human tissues, including heart and placenta.28Takenoshita S. Mogi A. Nagashima M. Yang K. Yagi K. Hanyu A. Nagamachi Y. Miyazono K. Hagiwara K. Characterization of the MADH2/Smad2 gene, a human Mad homolog responsible for the transforming growth factor-beta and activin signal transduction pathway.Genomics. 1998; 48: 1-11Crossref PubMed Scopus (45) Google Scholar There exists two mature Smad2 isoforms in human placenta: full-length Smad2, Smad2(FL), and Smad2 lacking exon 3, Smad2(Δexon3). Perhaps due to the specificity of the antibody and/or the protein extraction method,29Ueberham U. Lange P. Ueberham E. Brückner M.K. Hartlage-Rübsamen M. Pannicke T. Rohn S. Cross M. Arendt T. Smad2 isoforms are differentially expressed during mouse brain development and aging.Int. J. Dev. Neurosci. 2009; 27: 501-510Crossref PubMed Scopus (18) Google Scholar our previous work just detected the expression patterns of the Smad2(FL) in human placenta.7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar Despite that more than 90% sequences are similar between Smad2(FL) and Smad2(Δexon3), these two proteins have different expression patterns and distinguishing function features in several pathologies.29Ueberham U. Lange P. Ueberham E. Brückner M.K. Hartlage-Rübsamen M. Pannicke T. Rohn S. Cross M. Arendt T. Smad2 isoforms are differentially expressed during mouse brain development and aging.Int. J. Dev. Neurosci. 2009; 27: 501-510Crossref PubMed Scopus (18) Google Scholar,30Faure S. Lee M.A. Keller T. ten Dijke P. Whitman M. Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development.Development. 2000; 127: 2917-2931Crossref PubMed Google Scholar However, the expression pattern and function of these two Smad2 isoforms in severe PE (sPE) placenta remain unknown. In the present study, based on the evidence noted above, we have proposed that miR-18a regulated trophoblast cell function by targeting at one or more of the Smad proteins, which impaired the TGF-β signaling and led to the disease. To test our hypothesis, we used human trophoblast cell line HTR8/SVneo to investigate whether miR-18a attenuated the TGF-β signaling and inhibited the expression of Smad protein(s), the disturbance of which favors the disease development. With the use of quantitative real-time PCR technology, we examined pri-miR-18a and miR-18a expression levels in the control and sPE placentas. The levels of pri-miR-18a and miR-18a were significantly downregulated in the chorionic plates (Figures 1A and 1C ) but not in the basal plates (Figures 1B and 1D) of the sPE placentas. The expression pattern of miR-18a at different gestational stages of normal pregnancy was examined by quantitative real-time PCR. At least 3 samples were collected and analyzed in each gestational week. As shown in Figure 1E, the level of miR-18a during gestational weeks 8–10 was significantly higher than that at gestational weeks 6–7, and the level of miR-18a began to decrease after gestational week 10. The level of miR-18a at mid-term and term was significantly decreased compared to that at gestational weeks 6–7. We compared the expression levels of Smad2(FL), Smad2(Δexon3), and Smad3 in placentas from 13 sPE patients and 32 normal pregnant women, both at mRNA and protein levels. The mRNA expression of Smad3, Smad2(FL), and Smad2(Δexon3) had no obvious difference between sPE and control placentas in neither chorionic nor basal plates (Figures 2A, 2B, 3A , and 3B). The protein levels of Smad3 and Smad2(Δexon3) also exhibited no significant difference in sPE and normal placentas in either chorionic or basal plates (Figures 2C, 2D, 3C, and 3D). However, the Smad2(FL) protein level was increased in the chorionic plate, but not basal plates, of sPE placentas (Figures 3C and 3D). Interestingly, the p-Smad2(FL) protein level in the sPE placentas was also significantly increased in the chorionic plate but not the basal plates. There were no significant differences of the p-Smad2(Δexon3) between normal and sPE placentas either in the chorionic plate or in the basal plate (Figures 3E and 3F). p-Smad2(Δexon3) and Smad2(Δexon3) could not be detected in the Htr8/SVneo cells (Figure 3C–3F).Figure 3Differential Expression Patterns of Smad2 Isoforms and p-Smad2 Isoforms in Placentas Derived from sPE Patients and Normal Pregnant WomenShow full caption(A and B) Quantitative real-time PCR were performed to measure the expression of the Smad2(FL) and Smad2(Δexon3) in chorionic (A) and basal plates (B) of placentas derived from sPE patients (n = 13) and normal pregnant women (n = 32). (C–F) Western blotting was performed to measure the expression of Smad2(FL), Smad2(Δexon3), p-Smad2(FL), and p-Smad2(Δexon3) in chorionic (C and E) and basal plates (D and F) of placentas derived from sPE patients (n = 8) and normal pregnant women (n = 8). (G–J) The inverse correlation between the miR-18a and Smad2(FL) (or p-Smad2(FL)) expression in the placental chorionic (G and I) and basal (H and J) plates of the studied individuals was shown. Data are presented as mean ± SD. ∗p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Quantitative real-time PCR were performed to measure the expression of the Smad2(FL) and Smad2(Δexon3) in chorionic (A) and basal plates (B) of placentas derived from sPE patients (n = 13) and normal pregnant women (n = 32). (C–F) Western blotting was performed to measure the expression of Smad2(FL), Smad2(Δexon3), p-Smad2(FL), and p-Smad2(Δexon3) in chorionic (C and E) and basal plates (D and F) of placentas derived from sPE patients (n = 8) and normal pregnant women (n = 8). (G–J) The inverse correlation between the miR-18a and Smad2(FL) (or p-Smad2(FL)) expression in the placental chorionic (G and I) and basal (H and J) plates of the studied individuals was shown. Data are presented as mean ± SD. ∗p < 0.05. A correlation analysis of miR-18a concentration and Smad2(FL) (or p-Smad2(FL)) level was also performed accordingly. The data revealed an inverse correlation between miR-18a and Smad2(FL) (or p-Smad2(FL)) expression in the placental chorionic but not in the basal plates of the studied individuals (Figures 3G–3J). The antibody against Smad2 detected two bands on the western blots (Figure 3), which raises a question of whether it is due to an antibody-specificity issue or whether there are two variants of the protein. In addition, apart from Smad2(Δexon3) (composed of 437 amino acids), three other Smad2 variants (composed of 425 amino acids, 418 amino acids, and 414 amino acids, respectively) could also be retrieved in the NCBI database (https://www.ncbi.nlm.nih.gov). To verify that the lower band is Smad2(Δexon3), the corresponding protein spot was cut from the middle of the lanes and used for mass-spectrum analysis. As shown in Figure S1, five conserved peptides and one Smad2(Δexon3)-specific peptide were found, respectively. The five conserved peptide motifs were SLDGR, NATVEMTRR, RHIGR, MSFVK, and MSFVKGWGAEYR, respectively. The Smad2(Δexon3)-specific peptide motif was LDELEK. These results confirmed that there are two variants of Smad2, wild-type (WT) and mutant (MUT), and the MUT Smad2 isoform is Smad2(Δexon3). The localization of miR-18a and Smad2 in normal human placentas was examined by in situ hybridization and immunohistochemistry. Immunostaining for cytokeratin (CK)-8 was used as a marker for trophoblast cells (Figures 4E and 4F ). miR-18a and Smad2 were both localized in the in placental tissues, derived from gestational weeks 7–8, in similar patterns. They were predominantly localized in various subtypes of trophoblasts, including villous cytotrophoblast cells, syncytiotrophoblasts (Figures 4A and 4C), and interstitial trophoblasts cells invading the decidual stroma (Figures 4B and 4D). They were also weekly expressed in some villous mesenchymal cells (Figures 4A and 4C). No hybridization signals were detected in decidual cells (Figures 4B and 4D). In the negative control (NC) hybridization and immunochemistry without probe or primary antibody, no positive signals were observed (Figures 4G and 4H), showing the specificity of the experiments. We performed western blot analysis to detect syncytin-2 and human chorionic gonadotropin (hCG)-β of the primary syncytiotrophoblasts and cytotrophoblasts, which are markers for the cell type, which confirmed that syncytin-2 and hCG-β were present only in the primary cytotrophoblasts and syncytiotrophoblasts, respectively (Figure S2). We compared the expression level of miR-18a in three trophoblast cell lines (HTR8/SVneo, JEG-3, and B6-tert), as well as primary cultured cytotrophoblasts and syncytiotrophoblasts. We found that the HTR8/SVneo cells and the primary trophoblasts had comparable expression levels of miR-18a, whereas other two cell lines (JEG-3 and B6-tert) had a higher expression level than the primary cells (Figure S3), suggesting that HTR8/SVneo, rather than other two cell lines, had a miR-18a homeostasis similar to that in trophoblasts. Thus, the HTR8/SVneo cell line was selected as the experimental model to study the function of miR-18a in trophoblasts in vitro. As shown in Figures 5A and 5B , the transfection of miR-18a downregulated the expression of Smad2(FL) at both mRNA and protein levels, up to 76% and 54%, respectively. According to bioinformatics analysis, the seed sequence of miR-18a was complementary to 136 to 142 nt of 3ʹ UTR in Smad2(FL) mRNA. To further confirm that Smad2(FL) was the target of miR18a, the luciferase reporter construct carrying a 300-bp DNA fragment, including binding site (BD) of the 3ʹ UTR in human Smad2 mRNA (the vector was named BD-WT) or the point-mutated report construct (the vector was named BD-MUT) was transfected into HTR8/SVneo cells together with miR-18a mimics and thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK). 48 h after transfection, miR-18a mimics could evidently reduce the relative luciferase activity of the BD-WT construct by ≈35%, compared with scramble control, but could not affect the relative luciferase activity of the BD-MUT construct (Figures 5C and 5D). The effects of miR-18a on trophoblast cell invasion and proliferation were examined. Data from quantitative real-time PCR experiments showed that the level of miR-18a increased to ∼690-fold of that of the scramble control cells after miR-18a mimics transfection (Figure 6A). miR-18a inhibitor decreased the level of miR18a by ∼98% (Figure 6B). As shown in Figures 6C and 6D, overexpression of miR-18a in Htr8/SVneo cells significantly promoted cell invasiveness (Figure 6C). Conversely, inhibition of miR-18a could inhibit the cell-invasive potential (Figure 6D). In addition, miR-18a did not have an effect on cell growth (Figure 6E) or cell cycle (Figure 6F). We performed a rescue experiment by transfecting HTR8/SVneo cells with miR-18a together with pcDNA4-Smad2(FL). Interestingly, the invasion-promoting effect of miR-18a was inhibited by the overexpressed Smad2(FL) (Figures 6G, S4A, and S4B). With the consideration of the antagonistic potential of TGF-β signaling on trophoblast cell invasion, we investigated whether miR-18a could counteract the invasion suppression activity of TGF-β. The Htr8/SVneo cells were transfected with miR-18a mimics and then treated with recombinant TGF-β protein. We observed that TGF-β stimulated Smad2(FL) activation and inhibited the invasion in HTR8/SVneo cells (Figures 7A–7D). More importantly, transfection of miR-18a mimics inhibited the upregulation of p-Smad2(FL) and suppression of cell invasion by TGF-β (Figures 7A–7D). Since the first major study conducted by Pineles et al.,8Pineles B.L. Romero R. Montenegro D. Tarca A.L. Han Y.M. Kim Y.M. Draghici S. Espinoza J. Kusanovic J.P. Mittal P. et al.Distinct subsets of microRNAs are expressed differentially in the human placentas of patients with preeclampsia.Am. J. Obstet. Gynecol. 2007; 196: 261.e1-261.e6Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar more and more differentially expressed miRNAs have been identified in the placentas from PE patients. However, despite the wealth of studies that have suggested an association of certain miRNAs with PE, consensus has not yet been reached on which appears to be the key contributor to the pathogenesis of the condition and what the underlying mechanisms are. miR-18a is among the top 20 members of a list of differentially expressed miRNAs in PE placentas.31Sheikh A.M. Small H.Y. Currie G. Delles C. Systematic Review of Micro-RNA Expression in Pre-Eclampsia Identifies a Number of Common Pathways Associated with the Disease.PLoS ONE. 2016; 11: e0160808Crossref PubMed Scopus (51) Google Scholar With the consideration of the primary expression of miR-18a in various subtypes of placental trophoblasts,7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar it is likely that miR-18a may have functional roles in the regulation of trophoblast cell behaviors. Despite previous studies have demonstrated that this small RNA has biological functions in different cells,7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar,32Zhu X. Yang Y. Han T. Yin G. Gao P. Ni Y. Su X. Liu Y. Yao Y. Suppression of microRNA-18a expression inhibits invasion and promotes apoptosis of human trophoblast cells by targeting the estrogen receptor α gene.Mol. Med. Rep. 2015; 12: 2701-2706Crossref PubMed Scopus (24) Google Scholar, 33Krutilina R. Sun W. Sethuraman A. Brown M. Seagroves T.N. Pfeffer L.M. Ignatova T. Fan M. MicroRNA-18a inhibits hypoxia-inducible factor 1α activity and lung metastasis in basal breast cancers.Breast Cancer Res. 2014; 16: R78Crossref PubMed Scopus (72) Google Scholar, 34Chen X. Wang J. Cheng L. Lu M.P. miR-18a downregulates DICER1 and promotes proliferation and metastasis of nasopharyngeal carcinoma.Int. J. Clin. Exp. Med. 2014; 7: 847-855PubMed Google Scholar, 35Chen Y.J. Wu H. Zhu J.M. Li X.D. Luo S.W. Dong L. Liu T.T. Shen X.Z. MicroRNA-18a modulates P53 expression by targeting IRF2 in gastric cancer patients.J. Gastroenterol. Hepatol. 2016; 31: 155-163Crossref PubMed Scopus (40) Google Scholar, 36Song Y. Wang P. Zhao W. Yao Y. Liu X. Ma J. Xue Y. Liu Y. MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin.Exp. Cell Res. 2014; 324: 54-64Crossref PubMed Scopus (45) Google Scholar the existing results are conflicting. We proposed that such discrepancies might be due to the differences in microenvironments and regulatory networks in different kinds of cells. For instance, human placenta trophoblast cells share properties, such as active proliferation and invasion with tumor cells. However, these tumor-like behaviors in trophoblasts are tightly organized in unique temporal and spatial manners during pregnancy. In this study, we examined the expression profile of miR-18a in placentas at different gestational weeks and found that miR-18a was significantly upregulated during gestational weeks 8–10, followed by a decrease until term (Figure 1C), which is consistent with the previous results.37Morales-Prieto D.M. Chaiwangyen W. Ospina-Prieto S. Schneider U. Herrmann J. Gruhn B. Markert U.R. MicroRNA expression profiles of trophoblastic cells.Placenta. 2012; 33: 725-734Crossref PubMed Scopus (187) Google Scholar Since trophoblast cells are highly invasive at gestational weeks 8–10 during the whole gestation,38Lash G.E. Otun H.A. Innes B.A. Bulmer J.N. Searle R.F. Robson S.C. Low oxygen concentrations inhibit trophoblast cell invasion from early gestation placental explants via alterations in levels of the urokinase plasminogen activator system.Biol. Reprod. 2006; 74: 403-409Crossref PubMed Scopus (53) Google Scholar,39Genbacev O. Joslin R. Damsky C.H. Polliotti B.M. Fisher S.J. Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia.J. Clin. Invest. 1996; 97: 540-550Crossref PubMed Scopus (476) Google Scholar miR-18a may regulate trophoblast cell invasion. However, unlike the report in tumor cells, miR-18a substantially improved cell invasion (Figures 6C and 6D), but had no influence on cell growth and cell cycle in trophoblast cells (Figures 6E and 6F). These observations indicate that this small molecule has unique functions in placental trophoblast cells. Our results have shown that miR-18a promoted trophoblast invasion via targeting Smad2(FL): (1) The expression of Smad2(FL) was markedly upregulated in PE placentas, whereas miR-18a was decreased (Figures 1A, 1B, 3C, and 3D).7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar (2) miR-18a inhibited the expression of Smad2(FL) (Figures 5A and 5B).7Xu P. Zhao Y. Liu M. Wang Y. Wang H. Li Y.X. Zhu X. Yao Y. Wang H. Qiao J. et al.Variations of microRNAs in human placentas and plasma from preeclamptic pregnancy.Hypertension. 2014; 63: 1276-1284Crossref PubMed Scopus (156) Google Scholar (3) The luciferase reporter assay demonstrated the" @default.
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