Effect Of MiR-200a On The Proliferation And Apoptosis Of HEC-1B Cells By Targeting The Tumor Suppressor PTEN

Objective:The expression of hsa-miR-200a in HEC-1B cell line and innormal endometrial tissues were measured by real-time fluorescencequantitative PCR(FQ-PCR)． Synthetic hsa-miR-200a mimics and inhibitorwere trnsfected into HEC-1B cell line by lipofectamine.Cellulargrowth、proliferation capacity and apoptotic rate were assayed by themethyl thiazolyl tetrazolium (MTT) assay and floweytometry(FCM)． Bioinformatic analysis was used to predict targets ofhas-miR-200a on proliferation and apoptosis. Western blot was used tomeasure PTEN protein expression.EGFP fluorescence reporter vectoranalysis confirms that miR-200a targets PTEN in HEC-1B cells.Toclarify the mechanism of normal human reproductive process and diagnosisand treatment of diseases reproductive provided the basis.Methods:1.Cell culture and transfectionThe human endometrial adenocarcinoma cell line HEC-1B cells werecultured in DMEM/F12medium supplemented with10%fetal bovine serumat37℃and in a5%CO2atmosphere. Cells were plated at a density of5000 cells/well in96-well plates.Prepared one day after plating，The miR-200amimic-lipo2000mixture (final concentration of20nM), miR-200ainhibitor-lipo2000mixture (final concentration of100nM), and negativecontrols were added to the cells after a20-min incubation at roomtemperature.2.FQ-PCR analysis for mRNA and miRNA expressionTotal RNA from endometrial tissue or cells was extracted at48hpost-transfection using Trizol Reagent and was converted to cDNA byreverse transcription. Real-time quantitative PCR (FQ-PCR) wasperformed with SYBR green with U6as an internal reference fornormalization.3.MTT assayHEC-1B cells were seeded into96-well plates and transfected witha miR-200mimic, a miR-200inhibitor or negative controls the followingday. At24,48,72, and96h post-transfection, MTT (20μl) and DMSO(150μl) were added, and the absorbance of each well at a wavelength of570nm was detected with an ELISA plate reader. Each experiment wasrepeated three times.4.Cell cycle and apoptosis detection by flow cytometryTransfection was performed on cells in logarithmic growth phase.After48h, cells were rinsed with PBS and resuspended in ice-cold70%ethanol or PBS at a concentration of106cells/ml to prepare a single-cell suspension. The cell cycle distribution or apoptosis ratio of each samplewas determined by flow cytometry. Each experiment was repeated threetimes, and the mean was calculated.5.Bioinformatic analysisResults from the Pictar, MirGen, and Targetscan databases, whichpredict miRNA targets, were compared to find commonalities, and genefunction was analyzed using the Gene Ontology (GO) database.Proliferation or apoptosis-related genes were selected for furthercharacterization.6.Western blottingTotal cellular proteins were extracted by cell lysis, separated on an10%SDS-PAGE, transferred to PVDF, and incubated with anti-PTEN,anti-SON, and anti-PDCD4antibodies (1:50dilution) overnight at4℃. Thefollowing day, blots were incubated with a horseradishperoxidase-conjugated secondary antibody (1:500) for1h and visualizedby enhanced chemiluminescence (ECL).7.EGFP fluorescence reporter vector analysisEnhanced green fluorescent protein (EGFP) fluorescence reportervector analysis was performed by Saier Biotechnology, Inc.8.Statistical analysisSPSS17.0software was used for analysis of data. Statisticalsignificance was analyzed using the t test with statistical significance defined as P≤0.05.Results:1.Expression of miR-200aThe expression of miR-200a was detectable in the human EC cellline HEC-1B.2.Expression of miR-200a in each transfection groupThe expression of miR-200a in mimic-transfected cells wassignificantly higher than in the control, whereas the expression ininhibitor-transfected cells was significantly decreased (P<0.05). Nosignificant changes were observed in control and untreated cells.3.The effect of miR-200a expression on cell proliferationThe MTT assay was performed to measure the effect of miR-200aexpression on cell proliferation. The proliferation of HEC-1B cellstransfected with the miR-200a inhibitor was significantly lower thannormal cells and the negative control (P<0.05).The proliferation ofmimic-transfected HEC-1B cells exhibited no significant change (P>0.05).4.The effect of miR-200a expression on the cell cycleCell cycle distribution was analyzed by flow cytometry. Thepercentage of inhibitor-transfected cells in G1phase at48hpost-transfection was significantly higher than the blank control and thenegative control. The cell cycle distribution of mimic-transfected HEC-1Bcells exhibited no significant change (P>0.05). 5.The effect of miR-200a expression on apoptosisApoptosis was examined by flow cytometry. The percentage ofapoptotic inhibitor-transfected cells at48h post-transfection wassignificantly higher than the blank control and the negative control.Apoptosis among mimic-transfected HEC-1B cells was not significantlyaltered (P>0.05).6.Prediction of miR-200a targetsThrough bioinformatics, predicted by the database, get Son、Pdcd4、Pten as miR-200a targets, that related to the suppression of proliferationand the promotion of apoptosis were analyzed further.7.Negative regulation of PTEN by miR-200aThe mimic or inhibitor of miR-200a was transfected into HEC-1Bcells, and the expression of SON and PDCD4in HEC-1B cells did notchange significantly after transfection, whereas PTEN was down-regulatedin mimic-transfected cells and up-regulated in inhibitor-transfected cells.PTEN mRNA levels were not significantly different after transfection(P>0.05).8.miR-200a targets the3’ untranslated region of the PTEN mRNAThe expression of EGFP was lowest inpcDNA3/EGFP/PTEN+pri-200a-transfected cells, compared to the control(P<0.05), whereas no significant effect on EGFP expression was observedin MU-pcDNA3/EGFP/PTEN-transfected cells. Conclusion:1. The reduced expression of miR-200a can inhibit the proliferation ofHEC-1B cells and can promote apoptosis.2. Hsa-miR-200a plays an important role in the processes of proliferationand apoptosis in HEC-1B cells by regulating PTEN at the translationallevel.