5-AZA-CDR Effects On DNMTS/MBD2 Expression And Cell Growth In Human Choriocarcinoma Cell JEG-3

horionic carcinoma called choriocarcinoma for short,is an extremely common and highly malignant gestational trophoblastic neoplasia. Chemotherapy is the main clinical therapy , the cure rate can reach to 90%. However,in recent years,with the increase of multidrug-resistant chorioepithelioma which is difficult to cure, the clinic cure rate is decreased significantly.So before seeking for anti-chorioepithelioma drug,which is safe,effective,and with small toxic and side-effect,the most important thing is to clarify the mechanism of occurrence of choriocarcinoma.The pathological mechanism of chorioepielioma is complicate.The studies on genetic have shown a variety of oncogenes, tumor suppressor genes, abnormal expression of cellular transcription factors, and abnormal hormone levels associated with this disease [1,2].Present researches find the abnormal transcription of genes can induce by epigenetic, such as DNA methylation abnormalities which may be play an important role in the pathogenesis of choriocarcinoma.But it is not clear at present.DNA methylation is a reversible modification process,and the reversal of tumor cells can directly restore the function of certain tumor suppressor genes[3-6].Therefore,to study on point of view in DNA methyltransferase,cell proliferation,invasion and apoptosis can provide some fundamental experimental evidence for new strategy of the chorioepielioma treatment.We treated the choriocarcinoma JEG-3 cells with different concentrations(1,4,8,16,32μmol/L)of the DNA methyltransferase inhibitor 5-Aza-CdR.Then we used Real Time PCR and Western blotting to detect the change of mRNA and protein of DNA methyltransferases,DNA demethylase.Further more changes of cell morphology were observed under the microscope,and cell proliferation was detected by MTT.Flow Cytometry was used to detected JEG-3 cell apoptosis,and Transwell chamber measured the invasion of JEG-3 cells.Real Time PCR 5-Aza-CdR could significantly reduce the Dnmt1,Dnmt3a expression levels in a dose dependent manner,compared with the control degree of significant difference(P<0.05),and it could not affect the expression of (P> 0.05).(2) Western blotting results showed the same results as the Real Time PCR.5-Aza-CdR could significantly reduce the (P<0.05),and it could not affect the expression of (P>0.05).(3) After treatment JEG-3 cells with 5-Aza-CdR for 48 h,we observed by the microscope that the cells showed shrinkage,nuclear poly integrated block.Floating dead cells were significantly increased and cells grew badly.(4) MTT experiment results showed that different concentrations of 5-Aza-CdR could inhibit the proliferation of JEG-3 cells in a dose and time dependent manner.(5) The cell apoptosis was dected by flow cytometry.We found that the apoptosis rate was significantly increased with the multiplicative concentration of 5-Aza-CdR(P<0.01).(6) The invasion of JEG-3 was tested by Transwell small chamber. 5-Aza-CdR treatment led to depression of the cell invasiveness.There was a significant difference compared with the control group(P<0.01).5-Aza-CdR can inhibit the Dnmt1,Dnmt3a mRNA and protein expression remarkably,but it does not affect the Dnmt3b,Mbd2 mRNA and protein expression.5-Aza-CdR can also induce JEG-3 cell shrinkage,nuclear poly blocks,inhibit the cell proliferation and induce apoptosis and reduce the cell invasiveness.Dnmt1,Dnmt3a may play an important role in choriocarcinoma proliferation,invasion and apoptosis.