Expression Of Mmu-miR-96 In The Endometrium In Early Pregnancy And Its Regulation For Stromal Cells Apoptosis By Targeting Bcl2

Objective:Decidualization of endometrium stromal cells is an important feature of implantation and pregnancy.The molecular mechanism of decidualization remains unclear,especially micro RNAs regulation of this process.our project team previous ly study the micro RNA have demonstrated that mmu-miR-96 expression was higher at implantation sites in mouse uterus on day 5 of pregnancy compared with inter-implantation sites and was highest on day 1 of pregnancy by the micro RNA microarray analys is.Differential expression of mmu-miR-96 in mouse uterus during early pregnancy was to be verified by RT-PCR and in situ hybridization.Induction of artificial decidualization in vivo and in vitro was used to explore the possible impact on decidualization.We used database search to predict its possible target and lastly futher proved its target by Western blot and luciferase reporter,which provide experiment basis for futher study about the regulation of micro RNA on decidualization.Methods:(1)The result of miRNA microarray was demonsrtated by real-time fluorescence quantitative PCR.In situ hybridization detected the location of mmu-miR-96 of in endometrium of early pregnant mouse before and after the embryo implantation.(2)Induction of artific ial decidualization in vivo and in vitro modle was established to study the regulation role of mmu-miR-96 on endometrium decidual process.(3)Firstly isolation and culture of primary endometrial stromal cells transfected mmu-miR-96 inhibitor and mimics,and established in vivo model of artific ial induction decidua induced decidua,Expression of dtPRP and mmu-miR-96 detected by Real-time q PCR.Flow cytometry was used to detect the effect of mmu-miR-96 mimics on apoptosis and cell cycle.Secondly isolation and culture of primary decidual cells transfected mmu-miR-96 mimics,and detected apoptosis by flow cytometry as well.(4)MiRNAS target gene predic ition database was applied to search target genes of mmu-miR-96.We combined with the result of DGE analysis and found the predicted target gene.Western blot and luciferase reporter gene was choosed to further prove Bcl2 was inneed a target of mmu-miR-96.Results:(1)The result of real-time fluorescence quantitative PCR demonsrtated differential expression of mmu-miR-96 in mouse uterus during early pregnancy,which was consistent with the miRNA array.(2)In situ hybridization showed mmu-miR-96 was mainly located in the luminal epithelia on day 1,and weaker positive signals were observed in stromal cells on day 4,while its expression increased in stromal cells as well as embryo obviously on day 5 of pregnancy,and then mmu-miR-96 was observed widely expressed in second decidual zone(SDZ)on day 6,and expanded on day 7.(3)Compared with the control horn,the infused(oil)horn presented robust deciduoma,expression of mmu-miR-96 was obviously increased on the infused(oil)horn.(4)Apoptosis did not change after mmu-miR-96 inhibitor transfection,the level of dtPRP in the hormone treatment group increased significantly,while expression of dtPRP decreased after transfected with mmu-miR-96 mimic.Flow cytometry results suggested overexpression of mmu-miR-96 in stromal cells didn’t result in changes of cell cycle,while the rate of apoptotic cells was apparently increased following overexpression of mmu-miR-96.After the primary decidual cells were transfected with mmu-miR-96 mimics,the apoptosis rate of cells was observed.Consistently,excessive mmu-miR-96 would induce apoptosis of decidual cells(5)Three predicted target genes,namely Bcl2,Bcl212,Bcl213 was found through bioninformatics analysis and previously results.Endometrial stromal cells were transfected with mmu-miR-96 mimics,and Bcl2 protein level was obviously reduced when mmu-miR-96 was overexpressed in stromal cells.We also found overexpression of mmu-miR-96 luciferase activity was significantly reduced.Bcl2 was verified to be a target gene of mmu-miR-96 through above results.Conclusion:mmu-miR-96 may be involved in the regulation of apoptosis in decidual cells,which support subsequent development of fetus and gestation,moreover mmu-miR-96 may participate in apoptotic of stromal cells by regulating endogenous Bcl2,further play an improtant role in the process of embryo implantation.