Study On Effect And Molecular Mechanism Of TGF-β1 To The Proliferation And Invasion Of Choriocarcinoma JEG-3 Cell

The big family of transforming growth factor beta(TGF-β) includemany homoioplastic biologic cell factors. There are six subtypes at least(TGF-β1-6),but only three subtypes have been found in mammal(TGF-β1-3).Many kinds of normal and transforming cells can express TGF-β, such us activated T-cell, macrophage, trophiblast cell and so on. As the important factor related tophysiological accommodation,signal transduction TGF-βnot only plays a significant role in embryonic development, cell proliferation anddifferentiation, immune response and composition of extracellular matrix(ECM), but also closely correlated tothe genesis and development of tumor.Choriocarcinoma is a highly malignant tumor that originates from placenta trophiblast cell. Abnormal proliferation and invasion of trophblast cell lead to the development of choriocarcinoma.Now,increasing attention has beenpaid to the transforming pathway of TGF-β/Smads about the proliferation, invasion and metastasis of tumor.As an importantfactor,Smads can transmit signal to the cell nucleus, that is able to induce TGF-βnormal function.MMPs/TIMPs related to the degradation ofECM, take a part in invasion and metastasis of tumor cell. This kind of research on choriocarcinoma is fewreported. Therefore, we observe the effect of TGF-β1 on cell proliferation and invasion in choriocarcinoma, at the same time, we detectthe expressionsof Smad3-mRNA,Smad7mRNA, MMP-9protein, MMP-9mRNA, TIMP-1 protein and TIMP-1-mRNA in order to provide theoretic basis for molecular mechanism and new ideas for early treatment of choriocarcinoma in clinic.PARTⅠstudy on effect of TGF-β1 to the proliferation of JEG-3 cellObjectiveChoriocarcinoma JEG-3 cell were treated by TGF-β1. we use MTT to detect the proliferation of JEG-3 cell to observe the effectof TGF-β1on cell proliferation.MethodsThe study objective is immortalized choriocarcinoma JEG-3 Cell line.To digest JEG-3 cell in the period of logarithm growth to monoplast suspension with digestive juice containing 0.25% pan-creation and 0.02%EDTA. Make the concentration as 3×104/mL toculture in 96 well plates for 48 hours and then synchronize the JEG-3 cell with starvation for 24hours.Add recombinant TGF-β1 to make four concentrations(0,50,100,200ng/mL)and then incubated for 48 hours;Meanwhile, induce JEG-3 cell with TGF-β1100ng/mL,culturing for 0h, 12h, 24h, 48h respectively,eachgroup having 5 wells. Zero group and blank control group were all set up,each grouphaving 5 wells.The proliferation rate of cells was measured by MTT reductionassay.Results(1) With the concentration of TGF-β1 increasing, the JEG-3 cell proliferation activity were accelerated obviously in each group(P<0.05).(2) With the extention of TGF-β1 action time, the cell proliferationactivity were accelerated in each group(P<0.05).ConclusionExogenous TGF-β1 can promote the proliferation of JEG-3 choriocarcinoma cells, showing fine density-effect and time-effect relationship.PARTⅡstudy on effect of TGF-β1 to the expression of Smad3mRNA and Smad7mRNA in JEG-3 cellObjectiveWe add TGF-β1 with different density and action time to JEG-3cell RT-PCR were used to detect the expression of Smad3,Smad7mRNA,in order to discuss the mechanism of TGF-β1 on proliferation.Methods The cells were inoculated in 6 well plates at concentration of 1×105/mL.After cell synchronization, JEG-3 cell has been dividedinto the followinggroups. TGF-β1 concentration groups: 0, 100, 200ng/mL, incubating for 48 hours;TGF-β1 100ng/mL action time group:0h, 12h, 24h, 48h. Subsequently, the cell total RNA was extracted from these cells respectively to detected the expression of Smad3,7mRNA in each group through Reverse Transcription Poly-merase Chain Reaction (RT-PCR) methods.β-actin was the internal comparison.Results(1) After incubated with different TGF-β1 for 48 hours, along withTGF-β1 density increasing, the concentration of Smad3mRNA has raisedgradually(P<0.05),but Smad7mRNA has no significant change(P>0.05).(2) After incubated with TGF-β1 100ng/mL for different action time,along with action time extension,the concentration of Smad-3mRNA has raised gradually(P<0.05), but Smad7mRNA has no significant change(P>0.05).ConclusionChoriocarcinoma JEG-3 cell has no loss of Smad3,7mRNA. Within certain density and action time, TGF-β1 can promote the expressions ofSmad3mRNA and there are density-effect and time-effect relationship. Theexpression of Smad7mRNA has no response to the stimulation of TGF-β1.PARTⅢstudy on effect of TGF-β1 to the expression of MMP-9,TIMP-1protein and mRNA in JEG-3 cellObjectiveTreating JEG-3cell with TGF-β1,we use Western Blotting and RT-PCR to detect the expression of MMP-9, TIMP-1protein and mRNA in order to discuss the invasion mechanism of JEG-3 cell.MethodsJEG-3 cells with concentration of 1×105/mL were inoculated in 6 well plates and synchronized with starvation for 24 hours.And then add recombinant TGF-β1 to make three concentrations(0, 100, 200ng/mL) incubating for 48 hours. Meanwhile, TGF-β1 100ng/mL treated JEG-3 cell for 0h, 12h,24h, 48h,72h respectively, each grouphaving 5 wells.Thecell total protein and RNA were extracted from these cells respectively to determine the expression of MMP-9,TIMP-1 protein and mRNA in eachgroup with Western Blotting and RT-PCR.β-actin was the internalcomparison.Results(1) After incubated with TGF-β1 for 48 hours, the concentrate of MMP-9,TIMP-1 protein and mRNA have all increased gradually with TGF-β1density increasing(P<0.05).(2) After incubated with TGF-β1 100ng/mL for different action time,with the extention of TGF-β1 action time, the expressions of MMP-9,TIMP-1 protein and mRNA have all increased gradually(P<0.05).MMP-9/TIMP-1 and MMP-9mRNA/TIMP-1mRNA are bigger than one(P<0.05).ConclusionMMP-9/TIMP-1 has fine response to exogenous TGF-β1.Within certain density and action time, TGF-β1 can promote the expressionsof MMP-9,TIMP-1 protein and mRNA in Choriocarcinoma JEG-3 cell,and there aredensity-effect and time-effect relationship. MMP-9/TIMP-1 and MMP-9mRNA/TIMP-1mRNA are bigger than one. Maybe it is the mechanism which leads to the JEG-3cell invasion.