The Mechanism Research Of Cell Cycle Involved In Differentiation Of Endometrial Stromal Cells In Vitro

PartⅠThe establishment and identification of humanendometrium stroma cells (ESCs)decidualization in vitroObject:To establish the model of human endometrium stroma cells (ESCs) decidualizationculturing in vitro,and identify the efficiency of the decidualization model culturing in vitro.Methods:Human endometrium tissue was primary cultured.ESCs were treated by8-Br-cAMP and MPA after fourth passage.Immunocytochemical analysis of endometrialcells was conducted after the first passage.Keratin and Vimentin were used as markers ofepithelial cells and stroma cells.The ESCs morphology was observated by microscope andthe PRL level of cell cuiture fluid was detected by chemiluminescence duringdecidualization.Results:1.Vimentin espressed in cultured primary endometrial stroma cells cytoplasm,andKeratin unexpressed.2.ESCs morphology became round,cell and nucleus became biggerand the cell boundary became ambiguity from decidualizaton day 1 to day 4.3.The PRLlevel of cell cuiture fluid increased during the decidualization,and had no change in controlgroups.Conclusions:It is an ideally model to research decidualization that ESCs achieved decidualcells after treated four days by 8-Br-cAMP and MPA. PartⅡThe cell cycle and cell cycle array analysis of humanESCs decidualization in vitroObject:To study the differential cell cycle distribution of ESCs and differential expressionprofile of genes related to cell cycle during ESCs differentiation in vitro.Methods:Using 8-Br-cAMP and MPA to treat ESCs,D0 to D4 decidual ESCs andundecidual ESCs were analyzed cell cycle distribution by flow cytometrical analysis.Andthen D0 to D4 decidual ESCs and undecidual ESCs were analyzed by cell cycle gene array.Results:After decidulized for 0 hours (D0),24 hours (D1),48 hours (D2) and 96 hours(D4),the number of ESCs were 18.2%,8.51%,3.34% and 0.38% at S phase,5.7%,77.98%,84.88% and 88.63% at G0/G1 phase.Five genes were upregulated and eight genesdownregulated at least 2-fold between D2 and D0;four genes were upregulated and sevengenes downregulated at least 2-fold between D4 and D0;eleven genes were upregulatedand ten genes downregulated at least 2-fold between D4 and C4.Conclusions:During decidualization the number of ESCs decreased at S phase andincreased at G0/G1 phase.TIMP3,CDKN1C,CUL4B and CDKN2B were significantupregulated and CDK2,CCND1,CKS2,CDC6,CCNA2,CCNB1,CDC20 and CDC2were significant downregulated are involved in the mechanisms of ESCs cell cycleblockage during deciculazation. PartⅢThe micro-RNA array analysis of human ESCsdecidualization and undecidualization in vitro and prediction ofthe ability of some miRNAs to regulate ESCsdifferentiation-associated genesObject:To research micro-RNA variability espression between human ESCsdecidualization and undecidualization in vitro,and predict the ability of some miRNAs toregulate ESCs differentiation-associated genes.Methods:Using 8-Br-cAMP and MPA to treat ESCs,on treatment D4 decidual ESCs andundecidual ESCs were analyzed by microRNA array,and then we verified the results byreal-time PCR.The target genes about decidualization regulated by microRNA,and wereclassified by their function using bioinformatics method.Results:Microarry analysis show 16 miRNA genes were up-regulated in D4 and 33miRNA genes down-regulated in D4.To verify the accuracy of the microarray results,weused q-PCR to measure the expression levels ofhas-miR-27b,30c,143,101,181b,29b,30d,507,107,216,139,23a ,222,221 in C4 and D4,has-miR-27b,the expressionofhas-miR-30c,143,101,181b,29b,30d,507,23a ,222,221 was significantlydifference (P＜0.05) .On the basis of the target genes,the miRNAs annotated as havingdecidualization function might fall into 6 categories:(1) Those which might regulatedecidual specific transcription factors and DNA binding proteins;(2) Those which mightregulate extracellular matrix remodeling accompanied decidualization;(3) Those whichmight regulate growth factors,cytokines receptors and binding proteins;(4) Those whichmight promote ESCs exit the cell cycle and enter differentiation;(5) Those which mightregulate signal transduction during ESCs decidualization;(6) Those which might regulatedecidua-related enzyme metabolism.Conclusions:During decidualization of human endometrium,microRNA expresseddifferently and possibly played important role. PartⅣRegulation of cell cycle of ESCs during ESCsdifferentiation by hsa-miR-222 via CDKN1C/p57Object:To study the effect of hsa-miR-222 down-regulation by transfected hsa-miR-222inhibitors on ESCs cell cycle and CDKN1C/p57kip2 expression during ESCsdecidualization in vitro.Methods:We constructed 2'-O-Me-222-inhibitors oligonucleotide,their controls andFITC-conjugated control.ESCs were transfected with has-miR-222 inhibitors or with thecontrols.We observed the fluorescein expression by fluorescent microscope and assessedthe transfection efficiency.ESCs transfected were then decidualized with 8-Br-cAMP andMPA for 48 h or 0 h.We analyzed cell cycle by flow cytometrical analysis.And weconstructed and transfected pMIR-p57 vector with or without hsa-miR-222 inhibitor andthen analyzed firefly luciferase reporter activity by dual luciferase reporter gene assay andp57 protein expression by western blot analysis.Results:The number of ESCs treated with hsa-miR-222 inhibitors was decreased from10.93% to 6.25% at S phase,increased from 73.05% to 77.52% at G0/G1 phase.Duringdecidualization,decidualized for 48 h,induced ESCs treated with hsa-miR-222 inhibitorsdecreased from 7.83% to 3.34% in the S-phase population and increased from 74.94% to80.74% in the G0/G1 population.Western blotting confirmed a significant increase inCDKN1C/p57kip2 protein level in hsa-miR-222 inhibitor-treated ESCs as compared withthe control (P＜0.05) .Luciferase activity in ESCs transfected with p57 3'UTR-Luc andhsa-miR-222 inhibitor was increased of 93% compared with transfected with p573'UTR-Luc and negative control (p＜0.05)Conclusions:During decidualization the number of ESCs decreased at S phase,and afterdown-regulated hsa-miR-222 ESCs number of S phase decreased more and p57 proteinespression increased.So Hsa-miR-222 possibly regulated cell cycle of ESCs during ESCsdifferentiation via CDKN1 C/p57.