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postgraduate thesis: MicroRNA let-7a regulates integrin beta-3, vav3, and dicer to modulate trophoblast activities and hence embryo implantation

TitleMicroRNA let-7a regulates integrin beta-3, vav3, and dicer to modulate trophoblast activities and hence embryo implantation
Authors
Advisors
Issue Date2013
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Cheong, W. A. [張韻怡]. (2013). MicroRNA let-7a regulates integrin beta-3, vav3, and dicer to modulate trophoblast activities and hence embryo implantation. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5106512
AbstractMicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation.
DegreeDoctor of Philosophy
SubjectNon-coding RNA
Trophoblast
Ovum implantation
Small interfering RNA
Dept/ProgramObstetrics and Gynaecology
Persistent Identifierhttp://hdl.handle.net/10722/193411

 

DC FieldValueLanguage
dc.contributor.advisorYeung, WSB-
dc.contributor.advisorLee, CKF-
dc.contributor.authorCheong, Wan-yee, Ana-
dc.contributor.author張韻怡-
dc.date.accessioned2014-01-06T23:09:12Z-
dc.date.available2014-01-06T23:09:12Z-
dc.date.issued2013-
dc.identifier.citationCheong, W. A. [張韻怡]. (2013). MicroRNA let-7a regulates integrin beta-3, vav3, and dicer to modulate trophoblast activities and hence embryo implantation. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5106512-
dc.identifier.urihttp://hdl.handle.net/10722/193411-
dc.description.abstractMicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.subject.lcshNon-coding RNA-
dc.subject.lcshTrophoblast-
dc.subject.lcshOvum implantation-
dc.subject.lcshSmall interfering RNA-
dc.titleMicroRNA let-7a regulates integrin beta-3, vav3, and dicer to modulate trophoblast activities and hence embryo implantation-
dc.typePG_Thesis-
dc.identifier.hkulb5106512-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineObstetrics and Gynaecology-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5106512-
dc.date.hkucongregation2013-

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