[Correlation of netrin-1 expression with invasion of extra villous trophoblasts]

To investigate mechanism of netrin-1 regulating invasion of extra villous trophoblasts.

RT-PCR was used to detect six receptors expression including UNC5A, UNC5B, UNC5C, UNC5D, DCC and neogenin in extra villous trophoblast cell line TEV-1. The TEV-1 cells were cultured and devided into seven groups according to the concentration of netrin-1 adding into the medium, which include 10 microg/L, 50 microg/L, 100 microg/L, 500 microg/L, 1000 microg/L, 5000 microg/L and the control (the concentration of netrin-1 was 0 microg/L)groups. The proliferation and invasion of TEV-1 induced by netrin-1 were determined by CCK-8 assay and transwell invasion assay respectively.

(1) Only neogenin and UNC5B were found to be expressed on TEV-1 by RT-PCR method. (2) In CCK-8 proliferation assay, after 72 hours culture, the proliferation of TEV-1 were 1.55 +/- 0.29 in 10 microg/L, 1.72 +/- 0.31 in 50 microg/L, 2.15 +/- 0.35 in 100 microg/L, 1.42 +/- 0.25 in 500 microg/L, 1.50 +/- 0.27 in 1000 microg/L, and 1.38 +/- 0.23 in 5000 microg/L group, which were all higher than 1.00 +/- 0.16 in control group significantly (P < 0.05). (3) In matrigel invasion assay, after 6 hours culture, the number of the trans-membrane cells in various netrin-1 group, including 41 +/- 4 in 10 microg/L, 47 +/- 5 in 50 microg/L, 55 +/- 6 in 100 microg/L, 44 +/- 5 in 500 microg/L, 43 +/- 5 in 1000 microg/L and 42 +/- 5 in 5000 microg/L group, were all higher than 30 +/- 4 in control group with statistical significance (P < 0.05). (4) The fold changes of neogenin were 1.50 +/- 0.16 in 10 microg/L, 1.83 +/- 0.19 in 50 microg/L, 2.24 +/- 0.25 in 100 microg/L, 2.12 +/- 0.24 in 500 microg/L, 2.12 +/- 0.23 in 1000 microg/L and 2.13 +/- 0.23 in 5000 microg/L group, which were all higher than 1.00 +/- 0.11 in control group significantly (P < 0.05). There were significant difference between group 10 microg/L and 50 microg/L, group 50 microg/L and 100 microg/L (P < 0.05). There were no significant difference between group 100 microg/L and 500 microg/L, group 1000 microg/L and 5000 microg/L (P > 0.05). (5) The fold changes of UNC5B 1.09 +/- 0.11 in 10 microg/L, 1.47 +/- 0.14 in 50 microg/L, 1.61 +/- 0.16 in 100 microg/L, 1.85 +/- 0.19 in 500 microg/L, 2.21 +/- 0.21 in 1000 microg/L and 2.42 +/- 0.23 in 5000 microg/L group, were all higher significantly when compared with 1.00 +/- 0.07 in control group (P < 0.05). There were significant difference between all groups (P < 0.05).

Netrin-1 can promote the potential of proliferation and invasion of extravillous trophoblasts in vitro through its receptors including neogenin and UNC5B.