Homeobox Gene HLX1 Is A Regulator Of Hepatocyte Growth Factor Dependent Trophoblast Cell Invasion

【Background and Objective】Organogenesis of the human placenta involves certain key events including the development of the branched villous tree,vascularisation, syncytiotrophoblast membrane formation and extravillous trophoblast invasion into the maternal decidua and subsequent spiral artery remodelling, some investigators have proposed that many transcription factors that are important in the differentiation of the various cell types constituting the placenta. But very little or nothing is known about the upstream factors in the human placenta that modulate the expression of the regulatory factors and the downstream genes and pathways that are induced and/or repressed in response to the factors.Homeobox genes constitute an important family transcriptional regulators in placental development.HLXlalso known as HB24 were isolated from a human placental cDNA library and are predominantly expressed in the villous cytotrophoblast stem cell population of the developing placenta expression patterns suggest a role for HB24 in the control of cytotrophoblast differentiation. Rajaraman provided evidence of HLX regulation by growth factors and cytokines and established that HLX is an important regulator for signal transduction-mediated proliferation of human trophoblast cells. Previously, we observed Gene HLX1 silence could inhibit proliferation of HTR-8/SVneo cell and its invasion capacity as well. Hepatocyte growth factor (HGF) is a cytokine that is produced in the placental villous core and acts in a paracrine manner on trophoblasts that express the HGF receptor Met. Because HGF stimulates the invasion of many epithelial cell types, villous core HGF could regulate placental trophoblast invasion.In this study we postulate that HLX1 is a downstream effector gene of HGF stimulated signal transduction in the control of EVT invasion. To test this hypothesis, we have measured the effect of HGF stimulation on HLX1 mRNA production in cultured EVT cells. HLX1 gene expression was reduced by short interfering RNA (siRNA) oligonucleotides and the effect on trophoblast cell invasion was investigated.【Method】1. First trimester human EVT cell line HTR-8/SVneo was cultured and stimulated with various concentrations of HGF((0,10,20,50,100 ng/mL) for 24 hours.2. Transfection of siRNA using DharmaFECTM was conducted to silence homeobox gene HLX1 expression in HTR-8/SVneo cell.Then continued to stimulate the cells with 20 ng/mLHGF.3. Real-time quantitative PCR (qRT-PCR) and Western Blot were used to detect the expression of HLX1 mRNA and protein levels by siRNA, respectively.4. The expression of MMP-2 and MMP-9 in the cells were detected by quantitative zymography.5. Invasion assay:Transwell chambers were coated with Matrigel. HTR-8/SVneo cells were inoculated in the upper chambers at the same dose. RPMI-1640 media supplemented with 20% FCS was put in the lower chambers. After 24 hours incubation, the cells that had penetrated through to the bottom of the chamber were counted using microscope (40xfield).6. Statistical analysis:Experiments involving siRNA treatment were analysed by one-way analysis of variance (ANOVA) test, using SPSS11.0 for Windows. A probability value of<0.05 was considered significant.【Results】1. Compared to mock control,increasing HGF concentrations also increased HLX1 mRNA levels from 90% to 475% and a (156.76.4) %increased expresssion level of HLX1 protein with 20ng/ml HGF stimulation (both P< 0.01)2. HLX1 siRNA transfection resulted in a (54.57±0.31)% decrease of HLX1 mRNA level and a (68.44±2.48)% decrease expression level of protein (both P< 0.01) though in the presence of HGF stimulation.3. HGF stimulation resulted in a significant (394.6±2.9)% increase of MMP-2 in supernatant fluid but HLX1 down-regulation resulted in a (81.5±0.6)% decrease of MMP-2 though in the presence of HGF stimulation (both P< 0.01).4. HTR-8/Svneo cell invasion increased with a invasion number of (71±5), compared to the control group with a number of (50±3),P<0.01. Inhibition of HLX1 mRNA level suppressed cell invasive with a number of (20±4) compared to the MC group(43±3).【Conclusion】Our results suggest that In summary, we have shown in cultured trophoblast cells that a homeobox gene, HLX1, is a downstream effector gene of HGF, that HLX1 regulates placental cell invasion and that HGF acts, at least in part, through HLX1 to controlcell invasion capacities.