The Effect Of IL-6on Invasiveness Of Trophoblast Cells And Research Of The Related Signal Transduction Molecules

The establishment of successful human pregnancy is a complex processcontrolled by many factors including the formation and development of placenta. Asthe major component of placenta, trophoblast cells are critical for the implantation offetus, the development of placenta, the successful human pregnancy. During earlypregnancy trophoblast cells derive their origin from the ectoderm in embryo, and thendifferentiate into villous trophoblasts and extravillous trophoblasts (EVTs) followingthe development of pregnancy. Villous trophoblast cells for the chorionic villitransport nutrients to the fetus, while EVTs that invade into the decidual tissues tocomplete the reconstruction of spiral artery make enough maternal blood flow intointervillous space to maintain the normal physiological function of placenta.Trophoblast cells have similarities with the invasion of tumor cells. However, thedifference is that invasion of trophoblast cells is regulated with the strictly temporaland spatial limitations in an elegant way. Many studies have shown that invasiveinsufficiency during placentation contributes to a number of obstetrical complicationsincluding spontaneous abortion, fetal growth restriction and preeclampsia.Interleukin-6(IL-6) has a variety of biological function. The imbalance of IL-6was closely associated with the occurrence and recovery of many obstetric diseases,and plays an important role in the regulation of insiveness of tumor cells. As with IL-6, interleukin-11(IL-11) and leukaemia inhibitory factor (LIF) belongs to “IL-6cytokine super family”. IL-11and LIF have been identified as the critical factorsclosely associated with trophoblastic invasive process through JAK/STAT signaltransduction pathway. However, there was no unified conclusion about whether IL-6can promote the invasion of trophoblast cells. It has been widely known that a cell isinvasive through secreting protease with the virtue of hydrolysis. Matrixmetalloproteinases (MMPs), synthesized as latent proenzymes are capable to degrademost of extracellular matrix with the generation of their active forms. In the processof pregnancy, MMP-2and MMP-9contributing to the digestion of the maternalextracellular matrix (basic hurdle encountered by the invading trophoblast cells) werelosely associated with trophoblastic invasive process and have been known as thecritical protease during the implantation of placenta into the basement membrane ofmaternal uterus. However, the whole picture of regulatory mechanism of trophoblastinvasive process has not yet been fully elucidated, which involves complex cellularsignal transduction pathways, such as the JAK/STAT signal transduction pathway.Recent studies have revealed that STAT3may be one of the important upstreamfactors to regulate the expression of MMPs in breast cancer cells and melanoma cells.The human choriocarcinoma cell line JEG-3cells with similar molecules withtrophoblast cells can be used as the cellular models to rearch the invasiveness oftrhophoblast. In the present study, JEG-3cells were incubated with recombinanthuman IL-6and SD-1008(p-STAT3inhibitor) to analyze expression and activation ofMMPs for exploring the mechanism of IL-6inducing trophoblast invasion.ObjectThe aims of this study were to analyze the changes in mRNA and protein levelsof STAT3, p-STAT3, MMP-2, MMP-9in JEG-3cells following the incubation withIL-6or SD-1008by Real-time PCT and Western Blot assays and detect the possiblefunction of different concentration of IL-6on trophoblast invasiveness usingtranswell assays and MTT assays, in order to explore whether IL-6induce the invasion of trophoblast cells through up-regulating the expression or activation ofMMPs via STAT3signal pathway.Materials and methods1MaterialsJEG-3cell lines purchased from the Beijing Changsheng Biotechnology weremaintained with DMEM high sugar complete medium containing10%FBS,100U/mlpenicillin,100U/ml streptomycin at37°C under the moist atmosphere of air with5%CO2. JEG-3cells in exponential growth stage were used for all exposure experiments.2MethodsJEG-3cell lines were resuspended in complete DMEM-H medium andmaintained in humidified incubator with5%CO2at37°C constant temperaturechamber. Following the analysis of JEG-3cells growth curve, the exponential phasecells were seeded into6-well cell culture plate containing2.5ml of complete mediumat the density of1.5×106cells/well. After incubation for24h, JEG-3cells were madeinto three groups according their different bathes of cell cryopreservation: the controlgroup, the SD-1008(5μM) treatment group and the IL-6treatment group. Differentgroups of JEG-3cells were cultured for another24h and then used to obtain the totalRNA and proteins. Western Blot, Real-time polymerase chain reaction, transwellassay and MTT assay were used to investigate the changes in STAT3activity,expression of MMPs, invasion and proliferation of JEG-3choriocarcinoma cells.Based on the paired design, all the data from these assays were used to analyze theeffect of IL-6on JEG-3cells invasion and its molecular mechanism.3Statistics analysisStatistical analyses were performed using the SPSS version17.0software. Alldata were presented as mean±standard deviation. In the present study. Statistical differences of each two groups were calculated by comparing the meansusing Wilcoxon signed rank test when the data were out of normally distributed orStudent’s t-test if the data were normally distributed. Significance was consideredwhen P<0.05.Results1Effect of IL-6on invasion and proliferation of JEG-3cells in vitroTranswell assay was used to study the function of IL-6on invasion of JEG-3cells after incubation with different concentration of IL-6for24h. The resultsindicated that JEG-3cells treated with exogenous IL-6(10ng/ml) showed increasedinvasion (64.67±4.24) compared with control group (49.4±4.72)(P<0.05).Accordingly, IL-6was used at the10ng/ml concentration for subsequent experiments.MTT assay was used to detect the proliferation of JEG-3cells. The results showedthat there was no statistical difference between group administrated with IL-6and thecorresponding control group (P>0.05).2Effect of p-STAT3inhibitor on MMPs in JEG-3cellsIn order to analyse the induction of MMPs expression by JAK/STAT3signaling,SD-1008was used to inhibit the phosphorylation of STAT3. Incubation with SD-1008for24h led to reduced levels of p-STAT3(Tyr705)(0.06±0.02)(P<0.05) comparedwith control group (0.17±0.02) without the change in STAT3protein expression(P>0.05)). In the presence of SD-1008, gene expression of MMP-2decreasedsignificantly compared with control levels (0.42±0.24fold, P<0.05). Gene expressionlevels of MMP-9coincided with the MMP-2, which was significantly decreasedcompared with control group (0.48±0.21fold, P<0.05). The data of western blotassays in this study showed that changes in proMMP-2and proMMP-9proteinexpression were correlated with the mRNA levels in JEG-3cells after incubation for24h (P<0.05). 3Effect of IL-6on tyrosine phosphorylation of STAT3in JEG-3cellsIn consideration of inhibitory effect of p-STAT3inhibitor on MMPs gene andprotein expression in JEG-3cells, we also detected the phosphorylation of STAT3triggered by IL-6in JEG-3cells (starved for6h in serum-free medium) at differenttimes. However, the results showed that there was no change in levels ofphosphorylation of STAT3(Tyr705) in JEG-3cells following the incubation withIL-6(10ng/ml)(P>0.05).4Effect of IL-6on MMPs expression in JEG-3cellsReal-time PCR assays showed that there was no significant differernce in thelevels of MMP-2and MMP-9mRNA expression between the group administratedwith IL-6and the control group (P>0.05). In Western Blot assay, MMP-2primaryantibody to detect full length (proenzyme,72kDa) and cleaved (active enzyme,66kDa) MMP-2was used. Similarly, MMP-9primary antibody used in Western Blotassay is specific for pro (92kDa) and activated forms (84kDa) of MMP9. The resultsindicated that proMMP-2and proMMP-9protein levels had no change afterstimulation of IL-6compared with control group (P>0.05). In contrast, thestimulation of10ng/ml IL-6increased levels of activated form of MMP-2in JEG-3cells (0.78±0.18) compared with control group (0.5±0.11)(P<0.05). In this study,small amount of activated forms of MMP-9was detected which was hard to analysisthe difference between two groups.ConclusionsThis study suggested that trophoblastic invasion related factors (MMP-2andMMP-9) may be connected with the STAT3signaling. Moreover, IL-6play animportant role in inducing invasion of JEG-3cells via the activation of MMP-2,rather than inducing activation of STAT3to up-regulate the expression of MMPs.