N-Acetylglucosaminyltransferase Ⅲ Regulates Trophoblast Invasion Through N-Glycosylation α5β1 Integrin

ContextIn early pregnancy, trophoblast cells’attachment and invasion into maternal uterine tissue is essential for successful pregnancy and placentation. N-Acetyglucosaminyltransferase Ⅲ (GnT-Ⅲ) is one of the most important glycosyltransferases. GnT-Ⅲ and its product, bisecting N-acetylglucosamine (GlcNAc) branched N-glycan, are known to correlate with tumor suppression in most cancers. However, the expression of GnT-Ⅲ in different trimester human placenta and its role in the invasion of trophoblast cells have been not clear. In addition, changes in glycans on α5β1 integrin have been found to regulate cancer and EVT cell behavior.ObjectivesTo investigate the expression pattern of GnT-Ⅲ in different trimester human placenta, and explore the effects of GnT-Ⅲ knockdown on N-glycosylation status of α5β1 integrin and trophoblast cell migration and invasion in human choriocarcinoma cell line JEG-3. This may be help to find a new molecular marker for infertility and spontaneous abortion.Methods1. We used immunohistochemistry, quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunofluorescence to dectect the expression of GnT-III, and utilize biotinylated phaseolus vulgaris erythroagglutinin (PHA-E) lectin histochemistry, PHA-E lectin blotting and PHA-E lectin fluorescence to examine the expression of bisecting GlcNAc branched N-glycan in different trimester human placenta and various trophoblast cell lines.2. Small interfering RNA (siRNA) interference was used to knockdown the expression of GnT-Ⅲ in human choriocarcinoma cell line JEG-3. CCK-8, matrigel invasion, transwell migration, and extracellular matrix (ECM) protein adherence assays were used to detect the effect of GnT-Ⅲ knockdown on cell proliferation, invasion, migration and ECM protein adherence, respectively. Immunoprecipitation, western blot and qRT-PCR were used to analyze the effect of GnT-Ⅲ knockdown on α5β1 integrin. Western blotting was used to analyze the changes of some signaling pathways.Results1. It revealed that GnT-Ⅲ primarily expressed in the cytoplasm of placental villous cytotrophoblasts (CTBs) and syncytiotrophoblasts (STBs). The expression of GnT-Ⅲ in placental villi was decreased from first trimester to third trimester (P< 0.05).2. GnT-Ⅲ was strongly expressed in placental decidua which contained EVTs, and weak or no in those placental decidua without EVTs. Additionally, we observed that the staining intensity of GnT-Ⅲ was higher in the first trimester placental decidua compared to the third trimester decidua.3. GnT-Ⅲ products, bisecting GlcNAc branched N-glycans were mainly located outside of the trophoblasts layer in normal villi. They were expressed much more abundantly in the first trimester placental villi, and rarely expressed in the third trimester placental villi.4. The siRNA-mediated GnT-Ⅲ knockdown in human choriocarcinoma cell line JEG-3 decreased migration and invasion abilities, and reduced ECM protein adherence. GnT-Ⅲ knockdown reduced bisecting GlcNAc branched N-glycans of α5β1 integrin but not α5β1 integrin expression.5. GnT-Ⅲ knockdown down-regulated the expression of phosphorylated ERK and phosphorylated FAK.ConclusionThese results indicate that GnT-Ⅲ plays a key role in regulating the migration and invasion of trophoblast cells during human pregnancy. Alteration of bisecting GlcNAc branched N-glycans on α5β1 integrin may be involved in the regulation of this process. However, the detailed mechanism needs further exploration.