Effects Of HLA-G On Biological Behaviour Of JEG-3 Cell Line

Hypertensive disorders complicating pregnancy,which is a special desease involves to several systems and organs during pregnancy,seriously impairs the health of mather and infant,and is one of the major causes of the morbidity and mortality of infants and women in pregnancy and puerperium. Recently,it has been reported that HLA-G plays an important role in hypertensive disorders complicating pregnancy,but little is known about the pathogenesy of HLA-G in this desease.HLA-G,the nonclassical human major histocompatibility complex class I molecule,expresses mainly in extravillous cytotrophoblasts(EVCT) and JEG-3 cell line.HLA-G is considered to be correlated with the feto-maternal immune tolerance and prerequisite for successful pregnancy.The abnormal expression of HLA-G in EVCT may be associated with pathological pregnancy, such as hypertensive disorders complicating pregnancy.In our experiments,RNA interfence knocked down HLA-G expression in choriocarcinoma JEG-3 cells,and subsequently,western blot,RT-PCR,MTT, flow cytometric assessment and matrigel invasion assay were applied to explore the effect of HLA-G on JEG-3 cells proliferation,apoptosis and invasion.The results of our experiments suggest that decreased expression of HLA-G enhances trophocyte' s apoptosis and inhibits its proliferation and invasion.These experiments are divided into 2 parts,as following:(1) Inhibition of HLA-G expression in JEG-3 cell line by chemical synthesized siRNA;(2) Effects of HLA-G on biological behaviour of JEG-3 cell line Section 1 Inhibition of HLA-G expression in JEG-3 cell line by chemical synthesized siRNAObjective To obtain the effective siRNA which inhibits the expression of HLA-G in trophocyte cell line.Methods Human trophoblast-like cell line JEG-3 was used.The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA.Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and Western blot analysis.Results Expression of HLA-G mRNA and protein in JEG-3 cells was strongly inhibited by 80%- 90%after RNA interference by the three specific siRNA. Conclusions Inhibition of HLA-G expression in JEG-3 cell by RNAi will possibly provides a potent tool to investigate the function of HLA-G in these aspects,such as human fetal-maternal immunologic tolerance,the maintain of normal pregnancy and so on.Selection 2 Effects of HLA-G on biological behaviour of JEG-3 cell lineObjective To investigate the function of HLA-G in the growth and invasion of human trophoblast.Methods Human trophoblast-like cell line JEG-3 was used.The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA(siRNA).Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and Western Blot analysis.Changes of cell invasion,cycle,apoptosis and proliferation were respectively detected by Transwell wells test,flow cytometry,MTT assay.Results HLA-G siRNA transfection efficiency reached 80%- 90%. Expression of HLA-G mNNA and protein in JEG-3 cells was strongly inhibited by 80%- 90%after RNA interference.Inhibition of HLA-G expression decreased G2-M phase but increased S phase cells.The number of early apoptosis cells increased and the invasion ability of JEG-3 cells was significantly reduced.Conclusions HLA-G is possibly involved in the onset of hypertensive disorders complicating pregnancy respectively by regulating proliferation and invasion of trophoblast.