The MRNA Expression Of C-myc, TβRⅠ, TβRⅡ In Choriocarcin-oma JEG-3Induced By Transforming Growth Factor β1

Choriocarcinoma (choriocarcinoma CC) is a kind of asecondarytrophoblastic tumor (gestational trophoblastic tumor, GTT) which occurssecondary to normal or abnormal pregnancy.Choriocarcinoma can be dividedinto gestational choriocarcinoma (gestational choriocarcinoma, GC) and non-gestational choriocarcinoma (non-gestational choriocarcinoma,NGC).Transforming growth factor-β1(transforming growth factor-beta1, TGF-β1)which is a multifunctional cytokine polypeptide, become the most concernedcytokines in present study for its ability to regulate cell proliferation,differentiation, apoptosis, promoting extracellular matrix (ECM) andregulating the formation in embryonic development. TGF-β exerts itsbiological function by binding to its receptors, TβRⅠ and TβRⅡ, whichresult in the phosphorylation of receptor-regulated Smad proteins and adjustthe transcription of target gene in the nuclear. Any mutations of the componentof TβRⅠ,TβRⅡ or smad protein will cause the disorder of TGF-β/Smadsignaling pathway.Mitogen-activated protein kinase (mitogen-actived protein kinase, MAPK)pathway is one of the major intracellular signal transduction systems whichregulate a variety of biological responses. MAPKs consists of4distinctgroups, and p38MAPK play an important role In the process of cell malignanttransformation and tumor metastasis. Recent studies show that, p38MAPKpathway plays an important role in trophoblast invasion and the formation ofchoriocarcinoma, but the role of p38MAPK in mediatingTGF-β1signaltransduction process rarely reported. C-myc is known in many intracellularsignaling pathways as important downstream target genes, which plays animportant role in regulating cell proliferation, differentiation and apoptosis,and its overexpression can promote tumor cell growth[7]. Therefore, we usedMTT assay TGF-β1in different time JEG-3cells affect proliferation of choriocarcinoma, with TGF-β1and TGF receptor Ⅰinhibitor (LY364947)and p38MAPK inhibitor (SB203580) role JEG-after3cells to detect c-mycTβR Ⅰ, differential expression in JEG-3cells and TβR Ⅱ mRNA by qRT-PCRmethod to investigate choriocarcinoma in TGF-β1-mediated Smad and p38MAPK signal transduction mutual effect, which reveals deeper to malignanttransformation choriocarcinoma, the molecular mechanism of invasion andmetastasis.Objective:1.Pretreatment with TGF-β1choriocarcinoma JEG-3cells, the cellviability by MTT assay JEG-3cells to investigate the effect of TGF-β1choriocarcinoma cell activity2.Prior to joining TGF-β1with p38MAPK inhibitors and TGF-β receptorinhibitors, quantitative real-time RT-PCR assay to detect the level oftranscription TGF-β receptor.3.To observe the impact of TGF-β receptor1inhibitor and p38MAPKinhibitors on mRNA expression changes of c-myc in choriocarcinoma in JEG-3cell line.Methods:1.After reaching80%confluency, cells were trypsinized and cultured in96-well plates with an initial concentration of3×104/ml in1640containing10%FBS per well. After24hours’ culturing, the medium was changed to1640without FBS and cells were cultured for another12hours to ensure thecells synchronization. Wells were divided into7groups, and groups are asfollows: blank group, control group,1h group,2h group,6h group,12hgroup,24h group. TGF-β1(PeproTech Inc,USA) at the density of5ng/ml,was added to the plate except blank group and control group. Every specimenwas prepared in four replicates. By the time of1h, the medium of1h groupwas removed and180μL RPMI-1640supplemented with20μL of5ng/mLMTT (Sigma)in phosphate buffered saline was added to each well and theplate was incubated at37°C for4hours. Then, the medium was removed and150μl of dimethyl sulfoxide(DMSO, Sigma) per dish was added. After agitation for10minutes at room temprature, the absorbance of each group wasassayed at490nm with an enzyme-linked immunosorbent assay plate reader(bio-tek instruments). For2h group,6h group,12h group,24h group, thesame procedure was carried as1h group, by the time of2h,4h,6h and12hrespectively.2.JEG-3cells were incubated in6-well plate with an initial concentrationof5×104cells/ml for48hours. Wells were divided into8groups, and groupswere as follows: control group, TGF-β1group,1μM SB203580,3μMSB203580,1μM LY364947,3μM LY364947. Till cells reached approximately80%，cells in appropriate wells were pretreated with different concentrationsof TGF-β1receptor inhibitor and p38MAPK inhibitor cultured for2h, then1μM TGF-β1(PeproTech Inc,USA) were added into each well, except controlgroup, continuing incubated for2h.3.The JEG-3choriocarcinoma cells were treated with5ng/ml TGF-β1，andTGF-β receptor1inhibitor（LY364947）, p38MAPK inhibitors(SB203580)at the concentration of1μM,3μM respectively，mRNA levels of c-myc wasdetected by qRT-PCR.Results:1.JEG-3cells were treated with the same concentration of TGF-β1at5ng/ml, and then cellular proliferation was determined using the MTT assay atdifferent time. As showed in Fig1, JEG-3cells were not significantly affectedby the presence of TGF-β1at1and2h, compared with control group. Itsuggested that TGF-βdid not promote the proliferation of choriocarcinomabefore2h. By the time of6h, TGF-β1exhibits remarkable effect on JEG-3cell proliferation. And the viability of JEG-3cell proliferation was graduallydecreased by TGF-β1at12and24h.2.blocking p38MAPK pathway can modulate TβRⅡ transcriptional levelinduced by TGFβ-1in JEG-3cells.3.the mRNA level of c-myc in5ng/ml TGF-β1group increases,compared with control group （P <0.05）.Blockade of TGF-β pathway andp38MAPK pathway both can attenuated and inhibited the expressions of c- myc in a dose-dependent manner（P <0.05）Conclusions:1. blocking p38MAPK pathway can modulate TβRⅡ transcriptional levelinduced by TGFβ-1in JEG-3cells.2.C-myc is a downstream target gene of TGF β1/Smads pathway, and itsexpression level is regulated by the combination of TGFβ1and TGF-βreceptor1. Meanwhile, there’s an interaction between TGF-β1/smad signalpathway and p38MAPK pathway in choriocarcinoma JEG-3cell line.