Little is known about the effects of OSM on pregnancy, Sunitinib although OSM concentrations in the sera of pregnant women were found to be significantly higher than that in the sera of non pregnant women, throughout the preg nancy period. It is possible that OSM may affect the invasion and migration processes of the EVTs through various mechanisms, including its effect on EMT during early pregnancy. Our previous in vitro study demonstrated that OSM increases the invasion of EVTs in a first trimes ter EVT cell line. It has been reported that the loss of E cadherin with an increase of snail, which represses the transcription of E cadherin, is accompanied with an EMT in trophoblasts. The aim of the present study was to investigate the role of OSM on EVT migration and prolif eration with regard to its effects on the e pression of E cadherin, as a negative regulator of invasive behavior and related signaling pathways.
Methods Cell lines The EVT cell line HTR8 SVneo was kindly provided by Dr. Charles Graham. The cell line was produced by immortalization of HTR8 cells, an EVT cell line from primary e plant cultures of first trimester human placenta, with SV40. These cells e hibit markers of primary EVT cells, including the cytokeratins KRT7, KRT8, and KRT18, placental type alkaline phosphatase, high affinity PLAUR, human leukocyte antigen framework anti gen W6 32, HLA G, insulin like growth factor 2 mRNA, and a selective repertoire of integrins such as ITGA1, ITGA3, ITGA5, ITGAV, ITGB1, and ITGAVB3 B5. In the present study, HTR8 SVneo cells were used between passages 70 and 75.
Cell culture HTR8 SVneo cells were cultured in RPMI1640 containing 10% FBS. To analyze the effects of OSM on E cadherin in HTR8 SVneo cells, 107 cells were seeded in a 100 mm culture dish. After 24 h, the cells were treated with recombinant human OSM for the time indicated in the figure legends. Real time quantitative RT PCR analysis Total RNA was e tracted with TRIZOL reagent. The sequences of the primers used for real time PCR analysis for E cadherin and GAPDH were as follows E cadherin, GAPDH. cDNA synthesis cDNA was synthesized with 500 ng of RNA using the Superscript �� RT PCR System according to the manufactures recommenda tions. cDNA was diluted 1 2 prior to use in quantitative PCR. Quantitative TaqMan PCR PCR was performed in an ABI PRISM 7900HT Sequence Detection System in 384 well microtiter plates, with a final volume of 10 uL.
Optimum reaction conditions were established by using 5 ul of Universal Master Mi containing dNTPs, MgCl2, reac tion buffer and Ampli Taq Gold, 90 nM of primer and 250 nM fluorescence labeled TaqMan probe. Finally, 2 ul template cDNA was added to the reaction mi ture. The primer TaqMan probe combinations were designed Anacetrapib for each target sequence. The assay ID for the E cadherin probe was Hs01023894 m1.