[Correlation of netrin-1 expression with invasion of extra villous trophoblasts].
OBJECTIVE
To investigate mechanism of netrin-1 regulating invasion of extra villous trophoblasts.


METHODS
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.


RESULTS
(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).


CONCLUSIONS
Netrin-1 can promote the potential of proliferation and invasion of extravillous trophoblasts in vitro through its receptors including neogenin and UNC5B.
Yang
Yun
Y
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Wang
Yong
Y
DU
Yan
Y
Zhu
Jian-Wen
JW
Gao
Hui
H
Zou
Li
L
D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000008:administration & dosage / Q000494:pharmacology*; D000075386:Netrin Receptors; D000075388:Netrin-1; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014327:Trophoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000008:administration & dosage / Q000494:pharmacology*
D008565:Membrane Proteins; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D000075386:Netrin Receptors; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D025521:Tumor Suppressor Proteins; C532445:UNC5B protein, human; C091279:neogenin; D000075388:Netrin-1