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Regulation of muc1 expression by miRNA 199a in mouse endometrial epithelium during implantation
|關鍵字:||Implanation;著床;miRNA 199a;MUC1;miRNA 199a;MUC1||出版社:||動物科學系所||引用:||Abrahamsohn, P. A., T. M. Zorn and S. F. Oliveira. 1991. Extracellular matrix of the mouse endometrial during decidualization. Mem. Inst. Oswado. Cruz. 86: 23-25. Abrahamsohn, P. A. and T. M. Zorn. 1993. Implantation and decidualization in rodents. Exp. Zool. 266: 608-628. Andrew, S. K. 1998. Cytokines and implantation. Reprod. 3: 52-61. Aplin, J. D. 1997. Adhesion molecules in implantation. Rev. Reprod. 2: 84-93 Aplin, J. D., M. Meseguer, C. Simon, M. E. Ortiz, H. Croxatto and C. J. Jones. 2001. Muc1, glycans and the cell-surface barrier to embryo implantation. Biochem. Soc. Trans. 29: 153-156. Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116: 281-297. Bella, J., P. R. Kolatkar, C. W. Marior, J. M. Greve and M. G. Rossmann. 1998. 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Develop Growth & Differ. 31: 23-30. Zhang, B. and M. A. Farwell. 2008. microRNA: a new emerging class of players for disease diagnostic and gene therapy. J. Cell. Mol. Med. 12: 3-21.||摘要:||
胚胎著床為一複雜的過程，與囊胚直接和子宮上皮細胞接觸有關。於許多生物體中可發現微型RNA（miRNAs）的存在，其為21-24個核苷酸所構成的非編碼RNA。miRNAs被證實藉由調控基因表現參與許多細胞生理反應。本試驗中，欲探討著床過程中，著床相關之基因MUC1和miRNAs 199a於小鼠子宮上皮細胞之表現情形。MUC1為一種鑲嵌膜蛋白（integral membrane protein）表現於子宮及許多不同器官之腺體的上皮細胞中，並與胚胎著床過程中扮演重要的角色。減少子宮上皮細胞表面的MUC1表現為胚胎著床過程中所必需。試驗中進一步利用免疫組織化學染色法（Immunohistochemistry, IHC） 西方吸漬（western blot, WB） 即時聚合酶連鎖反應（Real-Time PCR） 轉染（Transfection） Dual-luciferase assay、免疫螢光染色法（Immunofluorescent, IFC） 及懷孕早期體外胚胎黏著試驗進行分析。於WB及IHC結果顯示，懷孕0.5天的小鼠子宮上皮細胞有較多MUC1表現，然而於4.5天時顯著下降。相反地，利用Real-Time PCR偵測懷孕小鼠子宮上皮細胞中miRNAs 199a的表現，發現於4.5天表量顯著下降。構築luciferase下游帶有MUC1-3’ UTR之載體，稱為pRL-MUC1，將其轉染至3T3細胞株中。利用 Dual-luciferase assay評估miRNAs 199a與MUC1-3’ UTR之結合情形，結果顯示，於轉染miRNAs 199a及pRL-MUC1組別中抑制luciferase的表現，然而，於轉染miRNAs 199a、pRL-MUC1與miRNAs 199a抑制子組別中可恢復luciferase的表現，因此認為miRNAs 199a藉由與MUC1-3’ UTR結合而調控MUC1之表現。另外，利用WB及IFC試驗證實，經由轉染後miRNAs 199a可抑制MUC1之表現。於懷孕早期體外胚胎黏著試驗中，發現轉染miRNAs 199a較無轉染miRNAs 199a之子宮內膜上皮細胞更具有使小鼠孵化的囊胚貼附於上皮細胞之能力，因此證實miRNAs 199a可直接調控MUC1之表現，並於懷孕早期具有抑制MUC1表現之功能。綜合上述，miRNAs 199a可能於成功懷孕過程中扮演重要之角色。
The embryo implantation process is complex, involving direct of the blastocyst with the luminal epithelium of receptive uterus. MicroRNAs (miRNAs) are 21-24 nucleotides non-coding RNA found in diverse organism. It has been shown that miRNAs participate in range of cellular process by means of regulating gene expression at post-transcriptional level. In this study, we have investigated miRNA 199a is spatiotemporally expressed in mouse uterus profiles during implantation coincident with expression of MUC1, a gene critical for implantation. MUC1 protein is an integral membrane protein that is expressed apically by simple secretory epithelium and glandular in many different organs which plays a fundamental role in embryo implantation. By means lossing of MUC1 on the surface of uterine epithelium cells are believed to be necessary for embryo implantation. In this study, an extensive analysis was performed by immunohistochemistry (IHC), western blot (WB), real-Time PCR, transfection, dual-luciferase assay, Immunofluorescent (IFC) and in vitro embryo adhesion assay during early pregnancy. The results generated from this present study indicate that MUC1 was much more on day 0.5 and decrease at day 4.5 of pregnancy base on observations from western blot and IHC and analyses, respectively. In contrast, miRNA 199a was expressed much more at day 4.5 of pregnancy after observed by real-time PCR. A reporter construct containing the MUC1-3' UTR located immediately downstream of luciferase, designated as pRL-MUC1, was transfected into 3T3 cell. The dual-luciferase assay was applied to evaluate the binding of miRNA 199a to the 3' UTR of MUC1. It was shown that the expression of luciferase was suppressed in the group of cells transfected with pRL-MUC1and miRNA 199a, and restored in the group of cells co-transfected with pRL-MUC1, miRNA199a and inhibitor of miRNA 199a, indicating the direct binding of miRNA 199a to the 3' UTR of MUC1 and regulatory effect of miRNA 199a on MUC1.By Using Western blot combined with, immunofluorescence staining and in vitro adhesion confirm that miRNA-199a can be down-regulation MUC1 expression with transfection. The hatching mouse blastocysts cultured on the endometrial epithelium which had been transfected with miRNA 199a precursor showed more adhesion ability than those cultured on the untransfected epithelium. We identified MUC1 as a direct target of miRNA 199a. By means, miRNA 199a down-regulated MUC1 expression at early pregnancy. The present data implicated that miRNA 199a might play a gateway role to a successful pregnancy.
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