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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

Overexpression and siRNA-mediated knockdown of MSX2 results in enhanced and reduced HTR8/SVneo cell invasion, respectively.(A) Overexpression of MSX2 induces invasion in HTR8/SVneo cells. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments (t-test). (c) Examination of MSX2 overexpression efficiency after transfection. And statistical assay of the results in (d) (t-test; *P < 0.05). (B) siRNA-mediated knockdown of MSX2 expression inhibits HTR8/SVneo cell invasion. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments. (t-test) (c) Examination of MSX2 knock down efficiency after siRNA transfection. And statistical assay of the results in (d) (t-test; **P < 0.01; n = 3). GAPDH was used as a loading control.
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pone.0153656.g002: Overexpression and siRNA-mediated knockdown of MSX2 results in enhanced and reduced HTR8/SVneo cell invasion, respectively.(A) Overexpression of MSX2 induces invasion in HTR8/SVneo cells. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments (t-test). (c) Examination of MSX2 overexpression efficiency after transfection. And statistical assay of the results in (d) (t-test; *P < 0.05). (B) siRNA-mediated knockdown of MSX2 expression inhibits HTR8/SVneo cell invasion. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments. (t-test) (c) Examination of MSX2 knock down efficiency after siRNA transfection. And statistical assay of the results in (d) (t-test; **P < 0.01; n = 3). GAPDH was used as a loading control.

Mentions: To investigate the role of MSX2 in trophoblast cell invasion, we utilized the human first trimester extravillous trophoblast cell line HTR8/SVneo. First, we successfully upregulated MSX2 expression(3.78±0.59 folds compared with MOCK) in these cells via transfection with a recombinant overexpression plasmid carrying the MSX2 gene (Fig 2Ac and 2Ad). The effects of MSX2 expression on motility and invasion of HTR8/SVneo cells were evaluated using Matrigel invasion assays. As shown in Fig 2Aa and 2Ab, overexpression of MSX2 resulted in marked increases in the invasive ability of HTR8/SVneo cells compared to the control. Conversely, transfection of HTR8/SVneo cells with a MSX2-specific siRNA resulted in suppression of MSX2 expression (0.47±0.09 folds compared with CON siRNA) and significantly diminished invasive ability, compared with the control cell population, as determined by Matrigel invasion assay(Fig 2B, 2Ba and 2Bb) and western blot analyses, respectively (Fig 2Bc and 2Bd).


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)

Overexpression and siRNA-mediated knockdown of MSX2 results in enhanced and reduced HTR8/SVneo cell invasion, respectively.(A) Overexpression of MSX2 induces invasion in HTR8/SVneo cells. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments (t-test). (c) Examination of MSX2 overexpression efficiency after transfection. And statistical assay of the results in (d) (t-test; *P < 0.05). (B) siRNA-mediated knockdown of MSX2 expression inhibits HTR8/SVneo cell invasion. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments. (t-test) (c) Examination of MSX2 knock down efficiency after siRNA transfection. And statistical assay of the results in (d) (t-test; **P < 0.01; n = 3). GAPDH was used as a loading control.
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Related In: Results  -  Collection

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pone.0153656.g002: Overexpression and siRNA-mediated knockdown of MSX2 results in enhanced and reduced HTR8/SVneo cell invasion, respectively.(A) Overexpression of MSX2 induces invasion in HTR8/SVneo cells. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments (t-test). (c) Examination of MSX2 overexpression efficiency after transfection. And statistical assay of the results in (d) (t-test; *P < 0.05). (B) siRNA-mediated knockdown of MSX2 expression inhibits HTR8/SVneo cell invasion. (a) Representative images of filters containing invaded cells from Matrigel invasion assays. (b) Statistical bar graphs showing the averaged results from three independent experiments. (t-test) (c) Examination of MSX2 knock down efficiency after siRNA transfection. And statistical assay of the results in (d) (t-test; **P < 0.01; n = 3). GAPDH was used as a loading control.
Mentions: To investigate the role of MSX2 in trophoblast cell invasion, we utilized the human first trimester extravillous trophoblast cell line HTR8/SVneo. First, we successfully upregulated MSX2 expression(3.78±0.59 folds compared with MOCK) in these cells via transfection with a recombinant overexpression plasmid carrying the MSX2 gene (Fig 2Ac and 2Ad). The effects of MSX2 expression on motility and invasion of HTR8/SVneo cells were evaluated using Matrigel invasion assays. As shown in Fig 2Aa and 2Ab, overexpression of MSX2 resulted in marked increases in the invasive ability of HTR8/SVneo cells compared to the control. Conversely, transfection of HTR8/SVneo cells with a MSX2-specific siRNA resulted in suppression of MSX2 expression (0.47±0.09 folds compared with CON siRNA) and significantly diminished invasive ability, compared with the control cell population, as determined by Matrigel invasion assay(Fig 2B, 2Ba and 2Bb) and western blot analyses, respectively (Fig 2Bc and 2Bd).

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