The Relationship Between TGF-β1and P38MAPK In The Occurrence And Development Of Choriocarcinoma JEG-3Cell

Choriocarcinoma, called CC for short, is highly malignant gestationaltrophoblastic. Forming from malignant transformation of trophoblastic stemcell, it could occur after abortion, ectopic pregnancy, hydatidiform mole,invasive mole and even normal pregnancy. During the early period ofChoriocarcinoma, which has a close relationship with excessive invasioncapacity of trophoblastic stem cell, blood route metastasis might occur so thatpatient’s life could be threatened. The occurrence of choriocarcinoma was leadby many factors, among which signaling pathway plays an important role.Nowadays the signal pathway of TGF-β is one of hot spots in researching ofpreventing and curing tumour. Transforming growth factor beta, TGF-β1, isresearched most in the filed of trophoblastic cell’s invasion involving theprocess of the mother’s body. TGF-β1acts as a key role in the malignantinvasion and transformation of gestational trophoblastic neoplasia throughTGF-β1signal pathway. P38mitogen-actived protein kinase (P38MAPK)which belongs to intracellular serine/threonine kinase is an intracellularprotein kinase existing human body. P38MAPK mainly involves cell growth,development, division, apoptosis and many physiological process. P38MAPKsignal pathway which is an important signal system to transmit extracellularstimulation in cell, works as a key role in the process of cell malignanttransformation and tumour infiltrating transformation. TGF-β1could stimulatedirectly the upstream kinase of P38MAPK signal pathway, so that stiulateindirectly P38MAPK signal pathway to involve the occurrence anddevelopment of tumour. In the research of many diseases’ pathogeneses,TGF-β1signal pathway and P38MAPK signal pathway have a certaininteractive effect, however, the report is rare whether the two signal pathwayshave interactive effect in choriocarcinama home and abroad. According to choriocarcinoma JEG-3model stimulated by low doseTGF-β1, using TGF-β1receptor inhibitor （LY364947） and p38MAPKinhibitor（SB203580） to block the two signal pathways, after the stimulationanalyzing the nuclear translocation of P38, the protein expressions of P38andphosphor-P38, the trial aims to reveal the interactive effect between TGF-β1signal pathway and P38MAPK signal pathway. The trial also states malignantinvasion and transforming molecular mechanism and calculates experimentalbases to provide a new target spot in clinical cure of choriocarcinoma.ObjectiveTo use TGF-β1with5ng/ml to pretreat choriocarcinoma JEG-3cell, tocheck nuclear translocation of P38after stimulation by the way ofImmunofluorescence analysis on the cell model, to test the protein expressionsof P38and phosphor-P38by the way of Western blotting, to discuss theinteractive effect of TGF-β1signal pathway and P38MAPK signal pathwayand the key procedures of the interactive effect to reveal the mechanism ofmalignant invasion and molecular transformation of choriocarcinoma.Methods1. the cultivation of cell and the establishment of cell mode:The object of study in the essay was the cell system of choriocarcinomawhich was brought from Institute of Basic Medical Sciences,Peking UnionMedical College. Completed substratum which concluded JEG-3andRPMI-1640with10%fetal bovine serum were cultivated in incubator with5%CO2at37C. when the cells reached approximately70%-80%, to go downto posterity at the proportion of0.25%trypsin to0.02%EDTA was1to3.Choosing JEG-3cells in logarithmic phase did experiment. Using TGF-β1with5ng/ml acted on JEG-3cells, establishing groups of comparison withoutstimulation. The groups of comparison included two dosages of TGF-β1receptor inhibitor (LY364947) and two dosages of P38MAPK inhibitor (SB203580).2. The process of nuclear transformation of P38after stimulation waschecked by immunofluorscence analysis. 3. The protein expressions of P38and phospo-P38were tested by Westernblotting.4. All experimental datum were analyzed by SPSS15.0software.Results1. Immunofluorescence analysis1.1TGF-β1receptor inhibitor (LY364947) affects on activation andnuclear transformation of P38in the JEG-3cell line.Immunofluorescence analysis indicated that stain of phospo-P38innucleus reduced after TGF-β1receptor inhibitor acted on JEG-3cells.According to the group with TGF-β1, the difference had statistic meaning (P＜0.05). Phospo-P38showed concentration dependent effect. That was to saystain of phospo-P38in nucleus decreased gradually as the concentrations ofthe TGF-β1receptor inhibitor increased (P＜0.05).1.2P38MAPK inhibitor (SB203580) affects on activation and nucleartransformation of P38in the JEG-3cell line.According to Immunofluorescence analysis, stain of phospo-P38innucleus reduced after P38MAPK inhibitor acted on JEG-3cells. According tothe group with TGF-β1, the difference had statistic meaning (P＜0.05).Phospo-P38showed concentration dependent effect. That was to say stain ofphospo-P38in nucleus decreased gradually with the increase of P38MAPKinhibitor’s concentration (P＜0.05).1. Western blotting detection2.1TGF-β1receptor inhibitor (LY364947) affects on protein expressionsof P38and phospo-P38in JEG-3cells.The trial indicated that protein expressions of P38and phospo-P38increased gradually after TGF-β1acted on JEG-3cells. The difference hadstatistic meaning comparing with the group without TGF-β1(P＜0.05); whileprotein expressions of P38and phospo-P38decreased gradually after addingTGF-β1receptor inhibitor. The difference had statistic meaning comparingwith the group with TGF-β1(P＜0.05); what’s more the density showeddependent effect, that was protein expressions of P38and phospo-P38 decreased gradually with the increase of the TGF-β1receptor inhibitor’sdensity.2.2P38MAPK inhibitor(SB203580) affects on protein expressions ofP38and phospo-P38in JEG-3cells.The trial indicated that protein expressions of P38and phospo-P38decreased gradually after P38MAPK inhibitor acted on JEG-3cells. Thedifference had statistic meaning comparing with the group with TGF-β1(P＜0.05); what’s more the density showed dependent effect, that was proteinexpressions of P38and phospo-P38decreased gradually with the increase ofP38MAPK inhibitor’s density.Conclusions1. Exogenous TGF-β1could promote P38’s nuclear transformation afteractivation in JEG-3cells, and could improve protein expressions of P38andphospo-P38in JEG-3cells.2.TGF-β1receptor inhibitor(LY364947) could reduce P38’s nucleartransformation after activation in JEG-3cells, and could decrease proteinexpressions of P38and phospo-P38in JEG-3cells, presenting goodconcentration dependant effect.3.P38MAPK inhibitor(SB203580) could reduce P38’s nucleartransformation after activation in JEG-3cells, and could decrease proteinexpressions of P38and phospo-P38in JEG-3cells, presenting goodconcentration dependant effect.4.There was a interactive effect between TGF-β1signal pathway andP38MAPK signal pathway in the malignant invasion of choriocarcinomaJEG-3cells.