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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

The effects of overexpression MSX2 on EMT markers in trophoblast cell lines.(A) Statistical analysis of mRNA expression levels in HTR8/SVneo cells after transfection with the indicated plasmids by quantitative reverse transcription (qRT)-PCR (Mann Whitney test; *P < 0.05). (B) HTR8/SVneo cells were transfected with the control or MSX2 expression plasmids and subjected to western blotting analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (C) (Mann Whitney test; *P < 0.05). (D) JEG-3 cells were transfected with the control or MSX2 expression plasmids and subjected to western blot analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (E) (Mann Whitney test; *P < 0.05).
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pone.0153656.g005: The effects of overexpression MSX2 on EMT markers in trophoblast cell lines.(A) Statistical analysis of mRNA expression levels in HTR8/SVneo cells after transfection with the indicated plasmids by quantitative reverse transcription (qRT)-PCR (Mann Whitney test; *P < 0.05). (B) HTR8/SVneo cells were transfected with the control or MSX2 expression plasmids and subjected to western blotting analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (C) (Mann Whitney test; *P < 0.05). (D) JEG-3 cells were transfected with the control or MSX2 expression plasmids and subjected to western blot analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (E) (Mann Whitney test; *P < 0.05).

Mentions: Previous studies demonstrated that MSX2 promotes EMT through activation of the Wnt/β-catenin signaling pathway in cancer cells [10, 15, 16, 22]. We therefore investigated the effects of differential MSX2 on the expression of β-catenin and vimentin (mesenchymal makers), and of E-cadherin (epithelial maker) in HTR8/SVneo and JEG-3 cells, respectively. qRT-PCR (Fig 5A) and western blot (Fig 5B and 5C) analyses indicated that overexpression of MSX2 resulted in reduced mRNA expression of E-cadherin and enhanced mRNA and protein expression of vimentin in HTR8/SVneo cells (P<0.05). Interestingly, overexpression of MSX2 also resulted in reduced mRNA expression but increased protein expression of β-catenin in HTR8/SVneo cells. Consistent with these findings, siRNA-mediated knockdown of MSX2 resulted in reduced mRNA (Fig 6A) and protein (Fig 6B and 6C) expression of vimentin in HTR8/SVneo cells(P<0.05). Furthermore, while the protein expression levels of β-catenin markedly reduced in cells treated with the MSX2 siRNA (P<0.05), these cells exhibited no changes in β-catenin mRNA levels(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)

The effects of overexpression MSX2 on EMT markers in trophoblast cell lines.(A) Statistical analysis of mRNA expression levels in HTR8/SVneo cells after transfection with the indicated plasmids by quantitative reverse transcription (qRT)-PCR (Mann Whitney test; *P < 0.05). (B) HTR8/SVneo cells were transfected with the control or MSX2 expression plasmids and subjected to western blotting analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (C) (Mann Whitney test; *P < 0.05). (D) JEG-3 cells were transfected with the control or MSX2 expression plasmids and subjected to western blot analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (E) (Mann Whitney test; *P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835074&req=5

pone.0153656.g005: The effects of overexpression MSX2 on EMT markers in trophoblast cell lines.(A) Statistical analysis of mRNA expression levels in HTR8/SVneo cells after transfection with the indicated plasmids by quantitative reverse transcription (qRT)-PCR (Mann Whitney test; *P < 0.05). (B) HTR8/SVneo cells were transfected with the control or MSX2 expression plasmids and subjected to western blotting analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (C) (Mann Whitney test; *P < 0.05). (D) JEG-3 cells were transfected with the control or MSX2 expression plasmids and subjected to western blot analysis using the indicated antibodies. And statistical assay from three independent experiments of the results in (E) (Mann Whitney test; *P < 0.05).
Mentions: Previous studies demonstrated that MSX2 promotes EMT through activation of the Wnt/β-catenin signaling pathway in cancer cells [10, 15, 16, 22]. We therefore investigated the effects of differential MSX2 on the expression of β-catenin and vimentin (mesenchymal makers), and of E-cadherin (epithelial maker) in HTR8/SVneo and JEG-3 cells, respectively. qRT-PCR (Fig 5A) and western blot (Fig 5B and 5C) analyses indicated that overexpression of MSX2 resulted in reduced mRNA expression of E-cadherin and enhanced mRNA and protein expression of vimentin in HTR8/SVneo cells (P<0.05). Interestingly, overexpression of MSX2 also resulted in reduced mRNA expression but increased protein expression of β-catenin in HTR8/SVneo cells. Consistent with these findings, siRNA-mediated knockdown of MSX2 resulted in reduced mRNA (Fig 6A) and protein (Fig 6B and 6C) expression of vimentin in HTR8/SVneo cells(P<0.05). Furthermore, while the protein expression levels of β-catenin markedly reduced in cells treated with the MSX2 siRNA (P<0.05), these cells exhibited no changes in β-catenin mRNA levels(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