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MSX2 Induces Trophoblast Invasion in Human Placenta.

Liang H, Zhang Q, Lu J, Yang G, Tian N, Wang X, Tan Y, Tan D - PLoS ONE (2016)

Bottom Line: Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin.Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells.Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Center, Chongqing Medical University, Chongqing, China.

ABSTRACT
Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

No MeSH data available.


Related in: MedlinePlus

Modulation of MSX2 expression in HTR8/SVneo cells influences the gelatinolytic activities of pro-MMP-2 and MMP-2.(A) Serum-free culture medium was harvested from cells transfected with the MSX2 overexpression vector and the MSX2 siRNA, as well as from the control cell populations, and subjected to gelatin zymography assay analysis. (B) Graphical representation of the zymographic results shown in A. (C) HTR8/SVneo cells transfected with plasmids or siRNA molecules were subjected to western blot analysis using the indicated antibodies. (D) Graphic depiction of the western blot results (three independent experiments). GAPDH was used as a loading control for western blot analyses (Mann Whitney test; *P < 0.05; n = 3).
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pone.0153656.g003: Modulation of MSX2 expression in HTR8/SVneo cells influences the gelatinolytic activities of pro-MMP-2 and MMP-2.(A) Serum-free culture medium was harvested from cells transfected with the MSX2 overexpression vector and the MSX2 siRNA, as well as from the control cell populations, and subjected to gelatin zymography assay analysis. (B) Graphical representation of the zymographic results shown in A. (C) HTR8/SVneo cells transfected with plasmids or siRNA molecules were subjected to western blot analysis using the indicated antibodies. (D) Graphic depiction of the western blot results (three independent experiments). GAPDH was used as a loading control for western blot analyses (Mann Whitney test; *P < 0.05; n = 3).

Mentions: Gelatinases (MMP-2 and MMP-9) play pivotal roles in the extracellular matrix remodeling during trophoblast invasion. As such, we investigated the effects of MSX2 expression on the activities of MMP-2 and MMP-9 in HTR8/SVneo cells. For these analyses, supernatant and total protein samples were harvest at 48 h after transfection with plasmids or siRNA molecules, and subjected to gelatin zymography assay analysis. Overexpression of MSX2 resulted in significant increases in pro-MMP-2, but not pro-MMP-9, activity in the supernatants of HTR8/SVneo cells, compared with the control (P<0.05). In contrast, the supernatant harvested from cells treated with the MSX2-specific siRNA exhibited prominently decreased pro-MMP-2, but not pro-MMP-9, activity (Fig 3A and 3B). Consistent with these findings, western blot analysis revealed that HTR8/SVneo cells transfected with the MSX2 plasmid and the MSX2-specific siRNA exhibited increased and decreased MMP-2 expression compared with the control cells, respectively, but no changes in MMP-9 expression (Fig 3C and 3D, P<0.05).


MSX2 Induces Trophoblast Invasion in Human Placenta.

Liang H, Zhang Q, Lu J, Yang G, Tian N, Wang X, Tan Y, Tan D - PLoS ONE (2016)

Modulation of MSX2 expression in HTR8/SVneo cells influences the gelatinolytic activities of pro-MMP-2 and MMP-2.(A) Serum-free culture medium was harvested from cells transfected with the MSX2 overexpression vector and the MSX2 siRNA, as well as from the control cell populations, and subjected to gelatin zymography assay analysis. (B) Graphical representation of the zymographic results shown in A. (C) HTR8/SVneo cells transfected with plasmids or siRNA molecules were subjected to western blot analysis using the indicated antibodies. (D) Graphic depiction of the western blot results (three independent experiments). GAPDH was used as a loading control for western blot analyses (Mann Whitney test; *P < 0.05; n = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835074&req=5

pone.0153656.g003: Modulation of MSX2 expression in HTR8/SVneo cells influences the gelatinolytic activities of pro-MMP-2 and MMP-2.(A) Serum-free culture medium was harvested from cells transfected with the MSX2 overexpression vector and the MSX2 siRNA, as well as from the control cell populations, and subjected to gelatin zymography assay analysis. (B) Graphical representation of the zymographic results shown in A. (C) HTR8/SVneo cells transfected with plasmids or siRNA molecules were subjected to western blot analysis using the indicated antibodies. (D) Graphic depiction of the western blot results (three independent experiments). GAPDH was used as a loading control for western blot analyses (Mann Whitney test; *P < 0.05; n = 3).
Mentions: Gelatinases (MMP-2 and MMP-9) play pivotal roles in the extracellular matrix remodeling during trophoblast invasion. As such, we investigated the effects of MSX2 expression on the activities of MMP-2 and MMP-9 in HTR8/SVneo cells. For these analyses, supernatant and total protein samples were harvest at 48 h after transfection with plasmids or siRNA molecules, and subjected to gelatin zymography assay analysis. Overexpression of MSX2 resulted in significant increases in pro-MMP-2, but not pro-MMP-9, activity in the supernatants of HTR8/SVneo cells, compared with the control (P<0.05). In contrast, the supernatant harvested from cells treated with the MSX2-specific siRNA exhibited prominently decreased pro-MMP-2, but not pro-MMP-9, activity (Fig 3A and 3B). Consistent with these findings, western blot analysis revealed that HTR8/SVneo cells transfected with the MSX2 plasmid and the MSX2-specific siRNA exhibited increased and decreased MMP-2 expression compared with the control cells, respectively, but no changes in MMP-9 expression (Fig 3C and 3D, P<0.05).

Bottom Line: Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin.Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells.Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Center, Chongqing Medical University, Chongqing, China.

ABSTRACT
Normal implantation depends on appropriate trophoblast growth and invasion. Inadequate trophoblast invasion results in pregnancy-related disorders, such as early miscarriage and pre-eclampsia, which are dangerous to both the mother and fetus. Msh Homeobox 2 (MSX2), a member of the MSX family of homeobox proteins, plays a significant role in the proliferation and differentiation of various cells and tissues, including ectodermal organs, teeth, and chondrocytes. Recently, MSX2 was found to play important roles in the invasion of cancer cells into adjacent tissues via the epithelial-mesenchymal transition (EMT). However, the role of MSX2 in trophoblastic invasion during placental development has yet to be explored. In the present study, we detected MSX2 expression in cytotrophoblast, syncytiotrophoblast, and extravillous cytotrophoblast cells of first or third trimester human placentas via immunohistochemistry analysis. Furthermore, we found that the in vitro invasive ability of HTR8/SVneo cells was enhanced by exogenous overexpression of MSX2, and that this effect was accompanied by increased protein expression of matrix metalloproteinase-2 (MMP-2), vimentin, and β-catenin. Conversely, treatment of HTR8/SVneo cells with MSX2-specific siRNAs resulted in decreased protein expression of MMP-2, vimentin, and β-catenin, and reduced invasion levels in a Matrigel invasion test. Notably, however, treatment with the MSX2 overexpression plasmid and the MSX2 siRNAs had no effect on the mRNA expression levels of β-catenin. Meanwhile, overexpression of MSX2 and treatment with the MSX2-specific siRNA resulted in decreased and increased E-cadherin expression, respectively, in JEG-3 cells. Lastly, the protein expression levels of MSX2 were significantly lower in human pre-eclamptic placental villi than in the matched control placentas. Collectively, our results suggest that MSX2 may induce human trophoblast cell invasion, and dysregulation of MSX2 expression may be associated with pre-eclampsia.

No MeSH data available.


Related in: MedlinePlus