1,721,137 research outputs found
Role of exosomal miR-141 in pro-metastatic tumor-stroma interactions in ovarian cancer
No full textMetastatic colonization is one of the critical steps in tumor metastasis. A pre-metastatic niche is required for metastatic colonization and is determined by tumor-stroma interactions, yet the mechanistic underpinnings remain incompletely understood. Previously, we showed that hsa-miR-141-3p (miR-141) is generally elevated and enhances anoikis resistance via targeting the KLF12/Sp1/survivin signaling cascade, facilitating metastatic cancer progression in ovarian cancer. Here, we report that Hsa-miR-141-3p (miR-141), an exosomal miRNA, is highly secreted by ovarian cancer cells and reprograms stromal fibroblasts into proinflammatory cancer-associated fibroblasts (CAFs), thereby facilitating metastatic colonization. A mechanistic study showed that miR-141 targeted Yes-associated protein (YAP1), a key effector of the Hippo pathway, promoting Tafazzin (TAZ), reducing nuclear YAP1/TAZ ratio and enhancing Tafazzin (TAZ)- and TEAD1-mediated transcriptional activation of the oncogenic factors GROα and EMMPRIN in stromal fibroblasts. Genetic inhibition of YAP1 induced a direct increase in transcription of GROα and EMMPRIN, in turn, forming an ideal pre-metastatic niche in tumor microenvironment (TME) under conditions of chronic inflammation favoring metastatic colonization and progression. Conversely, enforced expression of YAP1 markedly reduced the levels of GROα and EMMPRIN. Stromal-specific knockout (cKO) of Yap1 in a murine model shaped a GROα-enriched microenvironment and promoted tumor colonization in vivo, but this effect was reversed after Cxcr1/2 depletion in ovarian cancer cells. The YAP1/GROα correlation was demonstrated in clinical samples, highlighting the clinical relevance of this research and providing a potential therapeutic intervention for impeding premetastatic niche formation and metastatic progression of ovarian cancers.published_or_final_versionObstetrics and GynaecologyDoctoralDoctor of Philosoph
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Screening and characterization of pharmacologically active compounds regulating embryo implantation in vitro and in vivo
No full textAbstract of the thesis entitled
SCREENING AND CHARACTERIZATION OF PHARMACOLOGICALLY ACTIVE COMPOUNDS REGULATING EMBRYO IMPLANTATION
IN VITRO AND IN VIVO
Submitted by
CHEN Xian
for the degree of Doctor of Philosophy at The University of Hong Kong in March 2022
More than 25% of pregnancies are unintended. Current approaches to prevent pregnancy include physical or hormonal methods by inhibiting folliculogenesis, fertilization and embryo implantation. Hormonal contraceptive pills are widely used to avoid ovulation but are less effective once ovulation occurs. Other non-hormonal compounds that suppress embryo implantation can be potentially used for contraceptives are less explored. This study therefore aimed (1) to identify compounds that modulate spheroid attachment by screening the Library of Pharmacologically Active Compounds (LOPAC) in a high-throughput attachment assay, (2) to further identify compounds that suppress spheroid attachment on different human endometrial epithelial cells, and (3) to confirm the effects of such compounds in vivo using a mouse model.
A high-throughput in-vitro BeWo spheroids-Ishikawa endometrial epithelial cells co- culture model was established by quantifying fluorescent-labeled spheroids attachment in
a 96-well plate format. This model was used to screen the LOPAC and identify compounds that modulate spheroid attachment. The cytotoxicity and spheroid attachment in both endometrial Ishikawa and RL95-2 cells at different concentrations of selected compounds were studied. The number of implantation sites in pregnant ICR mice was studied after transcervical delivery of the compound into one uterine horn, and compared with the contralateral side transferred with solvent (5% DMSO) which was used as the control. Moreover, quantitative PCR was used to evaluate the expression of receptivity markers in the uterine epithelium of treated mice.
Of the 1280 compounds in LOPAC, 174 were identified that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 of these 174 compounds, Dihydroouabain (1, 3 and 10 μM), JS-K (10 μM) and Nemadipine-A (10 μM) significantly suppressed BeWo spheroid attachment in both Ishikawa and RL95-2 cells at the concentrations that are lower than their half-lethal concentrations (LC50). Dihydroouabain and Nemadipine-A also suppressed the BeWo spheroid attachment onto human primary endometrial epithelial cells collected at LH+7/8 days. A single transcervical transfer of Dihydroouabain (150 μg/kg and 15 μg/kg) or Nemadipine-A (100 μg/kg) suppressed embryo implantation sites significantly when compared with controls, while JS-K (6 μg/kg) was surprisingly found to increase implantation sites when compared with the control. The expressions of endometrial receptivity markers integrin αV (ITGAV) and mucin-1 (MUC1), but not β-catenin (CTNNB1), were significantly decreased in the uterus of ICR mice at 2.5 days post coitum (dpc) after treatment with Dihydroouabain, JS-K and Nemadipine-A. The suppression of embryo implantation by Dihydroouabain and Nemadipine-A was likely mediated through decreasing the mRNA expressions of ITGAV and MUC1.
Taken together, Dihryoouabain and Nemadipine-A suppressed spheroid attachment in vitro and embryo implantation in vivo. Detailed molecular and functional changes in mouse endometrium after treatment need to be investigated before the two compounds are used in the clinical setting. Also, the pharmacological role and doses of JS-K in regulating embryo implantation needs to be further explored. (Word count: 462)published_or_final_versionObstetrics and GynaecologyDoctoralDoctor of Philosoph
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
The regulatory role of adrenomedullin on macrophage activities and its association with the pathogenesis of tubal ectopic pregnancy
No full textTubal ectopic pregnancy (TEP) is one of the major causes of maternal morbidity and mortality in the first trimester of pregnancy. The most important predisposing condition of TEP is salpingitis. Adrenomedullin (ADM) is an immune-modulatory peptide with potent anti-inflammatory properties. Macrophage is the major immune cell population in the oviduct of TEP. They trigger the pro-inflammatory responses by secreting various pro-inflammatory cytokines. Both ADM insufficiency and macrophage accumulation in the fallopian tubes of TEP have been reported. The hypothesis of this study is that insufficiency of ADM expression in the fallopian tubes contributes to exacerbate pro-inflammatory responses of oviductal macrophages, consequently facilitates the tubal susceptibility for embryo-tubal implantation and the occurrence of TEP.
In this study, we included salpingitis as predisposing condition of TEP. Reduced ADM expression and macrophage infiltration, as well as elevated expression of implantation-related molecules such as E-cadherin, active β-catenin, leukemia inhibitory factor (LIF) and HomeoboxA (HoxA)-10 concomitantly took place in fallopian tubes of both salpingitis and TEP patients. In the oviductal macrophages from both salpingtis and TEP subjects, the expression of M1 macrophage marker inducible nitric oxide synthase (iNOS), but not M2 macrophage markers (CD163 and CD206), was observed. We also detected the expression of ADM receptors-calcitonin receptor like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2) in oviductal macrophages.
By using macrophages isolated from fallopian tubes of both salpingitis and TEP subjects, M1 human macrophages derived from human blood monocytes and monocytic cell line THP-1 derived macrophages, we demonstrated that ADM administration inhibited pro-inflammatory condition-induced upregulation of interleukin (IL)-6 and IL-8 in macrophages. The inhibitory effect of ADM on pro-inflammatory condition-induced IL-6 and IL-8 expression was further confirmed via CRLR knockdown by siRNA suppression. Moreover, we demonstrated that ADM treatment suppressed pro-inflammatory condition-induced activation of nuclear factor kappa B (NF-κB) signalling pathway by using THP-1 derived macrophages.
A JEG-3 trophoblastic spheroids-tubal epithelial cells (OE-E6/E7) co-culture model was used to study the protective role of ADM against tubal implantation. ADM effectively blocked the oviductal macrophage-derived conditioned medium-induced JEG-3 spheroids attachment onto tubal epithelial cells. Besides, ADM administration suppressed the oviductal macrophage-derived conditioned medium-induced upregulation of implantation-related molecules in tubal epithelial cells. This effect of ADM is possibly mediated by its inhibitory effect on IL-6 and IL-8 expression in human oviductal macrophages.
In conclusion, this study provided an explanation on the role of ADM in the pathogenesis of TEP by regulating macrophage activities. Our data revealed that ADM insufficiency in the fallopian tubes contributed to aggravated pro-inflammatory responses of oviductal macrophages by releasing implantation inducible pro-inflammatory cytokines, creating an adhesive and receptive tubal epithelium susceptible for embryo implantation with high levels of implantation-related molecules, further led to embryo-tubal implantation. The results of this study may indicate the possible use of ADM as a protective role against the occurrence of TEP. Further investigation is required to elucidate the detail actions of ADM in the pathogenesis of TEP.published_or_final_versionObstetrics and GynaecologyDoctoralDoctor of Philosoph
Microenvironment and its regulation on endometrial mesenchymal stromal/stem-like cells
No full textHuman endometrium undergoes cyclical shedding and remodeling in response to the steroid hormonal levels. The endometrial mesenchymal stromal/stem-like cells (eMSC) play crucial roles during endometrial regeneration and do not vary in frequency during the menstrual cycle. Stem cells often remain quiescent and undifferentiated when residing within the niche. The perivascular location of eMSC suggests that the endometrial stem cell niche is associated with blood vessels. However, the underlying mechanisms of eMSC responses to signals in the microenvironment are poorly understood. In this study, I hypothesized that endometrial stem cells can respond to specific molecules released by the microenvironment for the long-term maintenance of tissue hemostasis.
The first objective was to investigate the molecular mechanism of eMSC responses to myometrial secreted WNT5A. Among different WNT receptors, Frizzled -5 (FZD5) was abundantly expressed in CD140b+CD146+ eMSC when compared to unfractionated stromal cells. Co-expression of FZD5 and co-receptor LRP5 was confirmed in majority of eMSC population. Both gene silencing of FZD5 and blockage of LRP5 reduced the combination of eMSC with WNT5A and further inhibited the stimulatory effect of WNT5A on clonogenicity and phenotypic expression of eMSC. The WNT5A-FZD5-LRP5 complex regulated the self-renewal of eMSC through activating of WNT/β-catenin signaling pathway and a similar phenomenon was observed in gestational and postpartum mouse endometrial tissues.
The second objective was to evaluate the interplay between WNT/β-catenin and the Hippo signaling in eMSC by assessing the protein expression of their major components. The results showed that activities of Hippo signaling exhibited an antagonistic effect on WNT signaling in eMSC, and Hippo bioactive compounds such as CIL56 affected the proportion of eMSC, indicating a potential role of Hippo signaling on the cell fate of stem cells.
The third objective was to investigate the therapeutic potential of eMSC using the mouse endometrial injury model. Endometrial injury was induced using the electrocoagulation method and the regeneration of injured mouse endometrium with or without intrauterine eMSC transplantation were compared. Evaluation of morphological reconstruction revealed that eMSC transplantation increased the endometrial thickness, stimulated cell proliferation, promoted regeneration of endometrial compartments, relieved endometrial fibrosis and regulated inflammatory reactions in mouse endometrium. Fertility test confirmed the better endometrial receptivity of newly emerged endometrial lining with eMSC transplantation.
The fourth objective was to explore the role of interleukin-6 (IL-6) in endometrial stem cell regulation. The level of IL-6 secretion from endometrial stromal cells was high at menstruation and under hypoxic conditions. Functional assays revealed the stimulatory effects of exogenous IL-6 on self-renewal, proliferation and migration of eMSC in vitro, suggesting the involvement of IL-6 in endometrial regeneration. Activation of WNT/β-catenin signaling pathway was observed upon IL-6 treatment, suggesting the interplay of IL-6 and WNT signaling in modulation of eMSC biological activities. The therapeutic potential of IL-6 and WNT5A on endometrial regeneration was also uncovered by using the established mouse endometrial injury model.
In summary, niche components secrete regulatory molecules upon physiological signals. The responses of eMSC to these molecules result in the alterations of their properties for maintaining endometrial hemostasis involving intricate signaling networks.published_or_final_versionObstetrics and GynaecologyDoctoralDoctor of Philosoph
The role of Wnt-lin28/let7 signaling pathway in embryo implantation competency and trophoblast migration during early pregnancy
No full textEmbryo implantation consists of a series of complicated and coordinated events, including attachment of the embryo at the blastocyst stage to the endometrial epithelium, epithelial-mesenchymal transition (EMT) and penetration of the blastocyst through the surface of the endometrium. The outer layer of the penetrated blastocyst will then differentiate into the trophoblast cells leading eventually to the formation of the placenta. Implantation failure is a major cause of infertility, while uncontrolled implantation leads to ectopic pregnancy. Despite the importance of these events, our knowledge on the regulation of implantation and placentation differentiation is seriously limited.
Lethal-7 (let-7) is a family of miRNAs highly conserved among animal species. Aberrant expression of miRNA including mir-let-7 has been reported in blastocysts of infertile couples. Lin28 proteins is a natural repressor of mir-let-7 family. Our previous micro-array data has demonstrated that several members of the let-7 family were up-regulated in the dormant blastocysts when compared with the activated blastocysts. Furthermore, by performing signaling pathway analysis, Wnt signaling was shown to be one of the major regulators of mir-let-7 expression. Therefore, the hypothesis of this project was that Wnt/β-catenin signaling regulates embryo implantation competency, implantation accompanying embryonic trophectoderm EMT and uterine receptivity through lin28/let7 pathway.
The first objective examined the roles of Wnt/β-catenin-lin28a/let-7 pathway in regulating the implantation competency of mouse embryo/human embryo surrogate by using both in vivo and in vitro implantation model. It was demonstrated that Wnt/β-catenin signaling pathway stimulates lin28a expression, and thereby facilitates embryo implantation competency through down-regulating mir-let-7 expression.
The second objective investigated the roles of Wnt/β-catenin-lin28a/let7 pathway in regulating the EMT process of mouse embryo/human trophoblast during implantation. EMT was demonstrated to be occurred during mouse embryo outgrowth which is accompanied with Wnt-signaling activation and lin28a up-regulation. On the other hand, mir-let-7 overexpression hinders EMT and thus the implantation rate. Similar observation can be obtained in human trophoblast.
The third objective investigated the roles of mir-let-7 in regulating uterine receptivity. My results demonstrated that mir-let-7 expression is increased in peri-implantation mouse uterine epithelial cells when compared with the pre-implantation period. Maximal mir-let-7 expression can be detected at the implantation site. Exogenous mir-let-7 up-regulation induces both human and mouse uterine epithelial cell receptivity. However, in contrast to embryo/trophoblast, no lin28 expression can be detected in uterus, indicating that mir-let-7 in uterus is not regulated by lin28. Estrogen (E2) and progesterone (P4) was subsequently demonstrated to be the regulators of mir-let-7 expression during peri-implantation uterine epithelium.
In conclusion, this thesis demonstrated that Wnt/β-catenin suppresses mir-let-7 activity through lin28 stimulation to augment embryo implantation competency and induces EMT in embryo. In uterus, on the other hand, high level of mir-let-7 which is regulated by E2 and P4, is essential for establishing uterine receptivity. Results from this study provide the first demonstration on the biological role of Wnt/β-catenin-lin28a/let-7 pathway in implantation. Aberrant expression of let-7 in human blastocysts of infertile couples has been reported. In the long term, the result would provide the scientific basis for possible manipulation of let-7 on enhancing the implantation process.published_or_final_versionObstetrics and GynaecologyDoctoralDoctor of Philosoph
Investigation on the hCG-miR-125a-3p-Wnt axis through CTNNBIP1 in regulating endometrial receptivity and spheroid attachment
No full textSuccessful embryo implantation (EI) is a prerequisite to establish pregnancy. During embryo implantation, the blastocyst secretes human chorionic gonadotropin (hCG) that has been known to rescue corpus luteum, but its roles on EI remains elusive. Studies from this and other laboratory suggested that hCG regulates the expression of miR-125a-3p through the binding of LH/CG receptor (LHCGR) in the granulosa cells. MiR-125a-3p microRNA (miRNA) is a small single-stranded non-coding RNA molecule that is involved in oocyte maturation, and our recent finding suggested that miR-125a-3p binds to the 3’UTR of catenin beta-interacting protein 1 (CTNNBIP1) mRNA for RNA silencing and post-transcriptional regulation of gene expression. Importantly, CTNNBIP1 is an inhibitor of the Wnt signaling pathway that play important roles on EI. However, the exact role of CTNNBIP1 on EI remains elusive.
Yet, the Wnt signaling pathway is involved in many aspects of reproduction and fertility, including endometrial receptivity, EI, and placental development. This study aims to investigate the role of hCG on the expression of miR-125a-3p and the downstream target CTNNBIP1 on the regulation of Wnt-signaling molecules to spheroid attachment (mimicking EI process) using receptive (Ishikawa and RL95-2) and non-receptive (HEC1-B and AN3CA) human endometrial epithelial cells. MiRNA precursor and/or inhibitor were used to confirm the effect on spheroid attachment. CTNNBIP1 KO Ishikawa cells were established and the spheroid attachment rates are evaluated after 24 h hCG treatment.
It was found that the LHCGR transcript was detectable in receptive but not non-receptive human endometrial epithelial cell lines. HCG was found to induce miR-125a-3p and active--catenin to total -catenin expression in the receptive Ishikawa cells, and no change on CTNNBIP1 expression was found in all 4 cell lines. Interestingly, hCG significantly induce spheroid attachment in non-receptive cell lines (HEC1-B and AN3CA), whereas no further increase in the spheroid attachment in receptive cell lines. Transfection of miR-125a-3p precursor and inhibitor, significantly decrease and increase CTNNBIP1 mRNA, respectively, but no change in CTNNBIP1 protein expression was found. Neither the miR-125a-3p precursor nor inhibitor or CTNNBIP1 siRNA significantly affected total β-catenin expression. The transfection of miR-125a-3p precursor and inhibitor significantly increased and decreased the spheroid attachment rate onto Ishikawa cells. The CTNNBIP1 knockout cell lines were established using the Crispr/Cas9 system, and the Sanger sequences showed a770bp
deletion of the CTNNBIP1 gene when compared to the wild-type Ishikawa cells. The attachment rate was significantly increased in the CTNNBIP1 knockout cells. However, the knockout cells re-express CTNNBIP1 protein upon continuous passage.
In conclusion, hCG induces mir-125a-3p expression in the Ishikawa cells which may inhibit the suppressive effect of CTNNBIP1 on Wnt-signaling, the activated Wnt-signaling pathway increases active β-catenin expression and favor spheroid attachment. Although we have established the CTNNBIP1 KO cells, the stability of the cell line changes upon culture. A more in-depth investigation is needed to delineate the hCG-miR-125a-3p-Wnt axis on EI.published_or_final_versionObstetrics and GynaecologyMasterMaster of Philosoph
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