The irreversible loss of E cadherin expression emerges as a critical step driving epithelial mesenchymal transition in various human cancers. HTS The loss of E cadherin expression increases tumor invasiveness in vitro and in vivo and also increases the resistance of cancer cells to chemotherapeutic agents. Recent reports have implicated a crucial role for the miR 200 family in the regulation of E cadherin transcriptional repressors zinc finger E box binding homeobox 1 and zinc finger E box binding homeobox 2. In addition, the downregulation of DICER1 has been associated with the miR 200 family EMT pathway and tumor metasta sis, which indicates poorer prognosis. Here we presented for the first time a comprehensive analysis of miR 130 family and DICER1 expression in endometrial cancer tissues, compared with normal endo metrium.

In addition, with EC cells as experimental model we explored the mechanism and functional con sequences of dysregulation of some miRNAs, whose ex pression was linked to aberrant DNA methylation and histone modification and regulated the growth and inva sion of EC cells. Materials and Methods Cell culture and treatment The human endometrial cell lines Ishikawa and AN3CA were obtained from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. The cells were grown in Dulbeccos modified Eagles medium /F12 supplemented with 10% fetal bovine serum, 100 u/mL penicillin, and 100 ug/mL streptomycin in a humidified atmos phere of 5% CO2/95% air at 37 C. The cells were treated with 10 uM 5 Aza 2 deoxycytidine or 10 uM HDAC inhibitor Trichostatin A.

Cell transfection Cells were washed with PBS and transiently transfected with 100 nM pre miR 130b or anti miR 130b with their corresponding negative controls in Opti MEM using siPORT NeoFX transfection agent following the manufacturers protocol. Medium was replaced 8 h later. small interfering RNA expression vectors targeting DICER1 were transiently transfected into AN3CA and Ishikawa cells using lipofectamine 2000 following the manufacturers instructions. Quantitative real time PCR Fresh frozen EEC tissue samples and normal endometrial samples were obtained from patients at the Obstetrics and Gynecology Department of Shanghai First Peoples Hos pital, affiliated to Shanghai Jiao Tong University School of Medicine.

Following excision, tissue samples were imme diately snap frozen in liquid nitrogen and stored at ?80 C until RNA extraction. Total RNA was extracted from the tissues or cells using TRIzol RNA Isolation Reagents. The cDNA was generated using Prime Script RT reagent Kit Real time quantitative PCR of miRNAs was performed using TaqMan assay. The relative fold change was calculated based on the differences in Ct values between fold change 2 Ct. Three biological and technical replicates were done for each sample. All values were expressed Entinostat as mean standard deviation.