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Wan Yee Ana Cheong, Wei-Min Liu, Ronald Ting-Kai Pang, Kai-Fai Lee, William Shu-Biu Yeung, MicroRNA Let-7a Modulates Blastocyst Implantation of Mouse and Human via Its Action on Itgb3 and Vav3., Biology of Reproduction, Volume 87, Issue Suppl_1, 1 August 2012, Page 369, https://doi.org/10.1093/biolreprod/87.s1.369
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MicroRNA regulates protein expression by affecting the stability and/or translation of mRNAs. The microRNA let-7 is down-regulated during the pre-implantation mouse embryo development. Our preliminary results suggested that force-expression of let-7 hindered mouse blastocyst attachment and spreading on fibronectin-coated substratum in vitro and implantation in vivo. We hypothesized that let-7 affects implantation via its actions on implantation-related targets. In silico, the embryo-implantation-related integrin beta-3 (itgb3) and guanosine-exchange-factor vav3 were predicted targets of let-7. The interactions of let-7a with itgb3 and vav3 were confirmed by dual luciferase assay using the 3'-untranslated region reporter constructs of the target genes. Forced-expression of let-7a in the mouse blastocysts suppressed itgb3 and vav3 protein but not mRNA expression. Knockdown of itgb3 and vav3 suppressed blastocyst attachment in vitro. To test the relevance of these observations in human, a trophoblast(blastocyst surrogate)-endometrial cell co-culture model was used: the human trophoblast cells JAr formed spheroids in vitro and attached onto endometrial epithelial cells Ishikawa. Force-expression of let-7a in JAr cells suppressed the protein but not the transcript expression of itgb3 and vav3, supporting that let-7 regulated these gene expression post-transcriptionally. Upon treatment with anti-itgb3 antibody, the attachment rate of JAr spheroids onto Ishikawa monolayer and fibronectin was reduced by 50% at one-hour-post-treatment. The outgrowth area of the treated spheroids on fibronectin substratum was also reduced by half. Conversely, activating itgb3 by manganese chloride ameliorated the attachment rate of the treated JAr cells onto fibronectin. Moreover, in the mouse embryos, transfection of vav3 siRNA reduced their cell number by 50% and outgrowth area by three-fold: with a smaller and more polarized actin cytoskeleton distribution. This suggested that vav3 is conductive to cell proliferation and actin cytoskeletal reorganization to substantiate blastocyst outgrowth. We concluded that let-7 modulated implantation in mouse and human via its action on itgb3 and vav3. [The research work is partly supported by a GRF grant to WSBY].
