The Effects Of Homeobox Gene HLX1 Silence On Trophoblast Cell Proliferation And Invision

【Background and Objective】Homeobox genes are a large family of transcription factors that are essential for cell differentiation and morphological development.Of these,the HLX1 homeobox gene plays an important role in proliferation or differentiation in embryonic cell types and hematopoietic cell lineages and is proved to be an important factor for normal placentation.It has been reported that HLX1 mRNA expression is significantly reduced in idiopathic FGR-affected placentas compared with controls,and HLX1 protein is lately detected in cellular nuclei in the cytotrophoblast-derived cell lines HTR-8/SVneo,SGHPL-4,JEG-3,JAR and BeWo.In this article,we observed the suppression effects of homeobox gene HLX1 by small interference RNA(siRNA), investigated the consequential changes of biological performances of HTR-8/SVneo cell line and the possible role of HLX1 gene in the development of placental defect diseases such as idiopathic FGR.【Method】1.First trimester human EVT cell line HTR-8/SVneo was cultured.2.Reverse transcription-polymerase chain reaction(RT-PCR) and Western Blot were used to detect the expression of HLX1 mRNA and protein in HTR-8/SVneo cell, respectively.3.Based on the GenBank cDNA accession number NM₀21958,four independent HLX1 siRNA oligonucleotides were designed and obtained as siGENOME^TM SMARTpool^? siRNA.A negative siRNA,labelled with DY-547,was designed and obtained to be not specific for any known human gene.4.Transfection of siRNA using DharmaFECT^TM1 was conducted to silence homeobox gene HLX1 expression in HTR-8/SVneo cell.5.Flow cytometry(FCM) was used to assess and optimize siRNA transfection efficiency.6.Real-time quantitative PCR(qRT-PCR) and Western Blot were used to detect the depletion of HLX1 mRNA and protein levels by siRNA,respectively.7.Cell proliferation assay:Incubate HTR-8/SVneo cells at the same density in three groups overnight,then conduct transfection.Cell proliferation was assayed by methyl thiazolyl tetrazolium(MTT) after 24,48,72,96 hours of siRNA transfection respectively.Draw out their growth curves accordingly.8.Invasion assay:Transwell chambers were coated with Matrigel.HTR-8/SVneo cells were cultured at the same density in the three groups overnight and then conducted transfection.After 24 hours,cells were harvested respectively and 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(40×field).9.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.HLX1 mRNA and protein expression were testified in HTR-8/SVneo cell line.2.Transfection efficiency was up to(86.3±2.6)%at 24hs of Transfection after optimization.3.HLX1 siRNA transfection resulted in a significant(77.0±1.2)%decrease of HLX1 mRNA level and a(82.6±1.2)%decrease expression level of HLX1 protein (both P＜0.01).4.HLX1 siRNA resulted in a significant decrease of(58.1±4.4)%in trophoblast proliferation at 72hs of transfection(P＜0.01).5.HTR-8/SVneo cell invasion capacity was significantly suppressed,compared with two controls(P＜0.01).【Conclusion】Our results suggest that HLX1 siRNAs transfection effectively suppressed the expression of gene HLX1.Gene HLX1 silence could inhibit proliferation of HTR-8/SVneo cell and its invasion capacity as well.Aberrant HLX1 expression may impair both proliferation and invasion capacities of trophoblast cells,which is probably involved in the development of placental defect diseases such as FGR.