MicroRNA regulation of cyclooxygenase-2 during embryo implantation.
ABSTRACT: The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.
Project description:Vitrification is commonly used in the cryopreservation of mammalian blastocysts to overcome the temporal and spatial limitations of embryo transfer. Previous studies have shown that the implantation ability of vitrified blastocysts is impaired and that microRNAs (miRNAs) regulate the critical genes for embryo implantation. However, little information is available about the effect of vitrification on the miRNA transcriptome in blastocysts. In the present study, the miRNA transcriptomes in fresh and vitrified mouse blastocysts were analyzed by miRNA Taqman assay based method, and the results were validated using quantitative real-time PCR (qRT-PCR). Then, the differentially expressed miRNAs were assessed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 760 known mouse miRNAs were detected in the vitrified and fresh mouse blastocysts. Of these, the expression levels of five miRNAs differed significantly: in the vitrified blastocysts, four miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p) were upregulated, and one (mmu-miR-212-3p) was downregulated. The expression levels of all miRNAs measured by the miRNA Taqman assay based method and qRT-PCR were consistent. The four upregulated miRNAs were predicted to regulate 877 candidate target genes, and the downregulated miRNA was predicted to regulate 231 genes. The biological analysis further showed that the differentially expressed miRNAs mainly regulated the implantation of embryos. In conclusion, the results of our study showed that vitrification significantly altered the miRNA transcriptome in mouse blastocysts, which may decrease the implantation potential of vitrified blastocysts.
Project description:BACKGROUND: Delayed implantation is a developmental arrest at the blastocyst stage and a good model for embryo implantation. MicroRNAs (miRNAs) have been shown to be involved in mouse embryo implantation through regulating uterine gene expression. This study was to have an integrative analysis on global miRNA and mRNA expression in mouse uterus under delayed implantation and activation through Illumina sequencing. METHODOLOGY/PRINCIPAL FINDINGS: By deep sequencing and analysis, we found that there are 20 miRNAs up-regulated and 42 miRNAs down-regulated at least 1.2 folds, and 268 genes up-regulated and 295 genes down-regulated at least 2 folds under activation compared to delayed implantation, respectively. Many different forms of editing in mature miRNAs are detected. The percentage of editing at positions 4 and 5 of mature miRNAs is significantly higher under delayed implantation than under activation. Although the number of miR-21 reference sequence under activation is slightly lower than that under delayed implantation, the total level of miR-21 under activation is higher than that under delayed implantation. Six novel miRNAs are predicted and confirmed. The target genes of significantly up-regulated miRNAs under activation are significantly enriched. CONCLUSIONS: miRNA and mRNA expression patterns are closely related. The target genes of up-regulated miRNAs are significantly enriched. A high level of editing at positions 4 and 5 of mature miRNAs is detected under delayed implantation than under activation. Our data should be valuable for future study on delayed implantation.
Project description:BACKGROUND:Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8?weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed. RESULTS:Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity. CONCLUSIONS:This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.
Project description:Human preimplantation embryo development is susceptible to high rates of early embryo wastage. We determined the miR-21 expression of extracellular vesicles (EVs) in fertilized eggs and embryos of varying stages and their response to miR-21 microinjection. Sexually mature female and male mice were mated. Next, the expression and immunohistochemistry intensity of surface markers (CD9 and CD63) of EVs were detected in pregnant and non-pregnant mice. Exosomes were co-cultured with embryos for detection of blastocyst formation rate, and embryo apoptosis. Moreover, the expressions of Bcl-2 associated X protein (Bax), B cell lymphoma 2 (Bcl-2), and octamer-binding transcription factor-4 (Oct4) were determined. Finally, we detected miR-21 expression in EVs of uterus in pregnant mice, in embryos after embryo implantation and after embryo co-cultured with exosomes in uterine luminal fluid. MiR-21 was up-regulated in EVs of uterus, and higher immunohistochemistry intensity of CD9 and CD63, suggesting more EVs secreted in uterine luminal fluid in pregnant mice. After microinjection, miR-21 inhibitor suppresses embryo development of mice. Moreover, embryos co-cultured with exosomes display higher blastocyst formation rate, reduced apoptotic rate of embryos in pregnant mice. In addition, miR-21 was down-regulated with the development of embryos after embryo implantation, while miR-21 expression in embryos was up-regulated by exosomes in uterine luminal fluid in the pregnant mice. Increased miR-21 expression in EVs of uterus and increased miR-21 expression after implantation, which indicate the key role in the growth of fertilized eggs and embryo development in mice.
Project description:Successful embryo implantation requires functional luminal epithelia to establish uterine receptivity and blastocyst-uterine adhesion. During the configuration of uterine receptivity from prereceptive phase, the luminal epithelium undergoes dynamic membrane reorganization and depolarization. This timely regulated epithelial membrane maturation and precisely maintained epithelial integrity are critical for embryo implantation in both humans and mice. However, it remained largely unexplored with respect to potential signaling cascades governing this functional epithelial transformation prior to implantation. Using multiple genetic and cellular approaches combined with uterine conditional Rac1 deletion mouse model, we demonstrated herein that Rac1, a small GTPase, is spatiotemporally expressed in the periimplantation uterus, and uterine depletion of Rac1 induces premature decrease of epithelial apical-basal polarity and defective junction remodeling, leading to disrupted uterine receptivity and implantation failure. Further investigations identified Pak1-ERM as a downstream signaling cascade upon Rac1 activation in the luminal epithelium necessary for uterine receptivity. In addition, we also demonstrated that Rac1 via P38 MAPK signaling ensures timely epithelial apoptotic death at postimplantation. Besides uncovering a potentially important molecule machinery governing uterine luminal integrity for embryo implantation, our finding has high clinical relevance, because Rac1 is essential for normal endometrial functions in women.
Project description:Bladder outlet obstruction (BOO) leads to lower urinary tract symptoms (LUTS) and urodynamic changes of the bladder function. Previously we identified microRNA (miRNA) and mRNA expression profiles associated with different states of BOO-induced LUTD in human patients. Bladder wall remodeling resulting from obstruction is widely studied in animal models of experimentally-induced partial BOO (pBOO). Here we determined the expression profiles of miRNAs and selected mRNAs in pBOO mice and compared the observed changes to human patients. Similar to results from human patients, we observed a down-regulation of smooth muscle-associated miRNAs mmu-miR-1, mmu-miR-143, mmu-miR-145, mmu-miR-486 and mmu-miR-133a in pBOO mouse bladders. Pro-fibrotic miRNAs mmu-miR-142-3p and mmu-miR-21 were up-regulated, and anti-fibrotic miRNA mmu-miR-29c was down-regulated. Pathway analysis in human BOO patients identified TNF-alpha as the top upstream regulator. Although there was evidence of hypertrophic changes in pBOO mice, contrary to human data, we observed no regulation of TNF-responsive genes in the mouse model. Experimentally-induced pBOO in mice led to significant gene expression changes, including alteration of pro-fibrotic mRNAs and miRNAs resembling human BOO patients. Gene expression changes were also validated in a mouse model of bladder inflammation. Lack of evidence of TNF-alpha-induced miRNA and mRNA regulation might indicate a different pathophysiological mechanism of organ remodeling in pBOO model compared to the human disease.
Project description:Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.
Project description:Although microRNAs (miRNAs) play an important role in liver homeostasis, the extent to which they can be altered by Toxoplasma gondii infection is unknown. Here, we utilized small RNA sequencing and bioinformatic analyses to characterize miRNA expression profiles in the liver of domestic cats at 7 days after oral infection with T. gondii (Type II) strain. A total of 384 miRNAs were identified and 82 were differentially expressed, of which 33 were up-regulated and 49 down-regulated. Also, 5690 predicted host gene targets for the differentially expressed miRNAs were identified using the bioinformatic algorithm miRanda. Gene ontology analysis revealed that the predicted gene targets of the dysregulated miRNAs were significantly enriched in apoptosis. Kyoto Encyclopedia of Genes and Genomes analysis showed that the predicted gene targets were involved in several pathways, including acute myeloid leukemia, central carbon metabolism in cancer, choline metabolism in cancer, estrogen signaling pathway, fatty acid degradation, lysosome, nucleotide excision repair, progesterone-mediated oocyte maturation, and VEGF signaling pathway. The expression level of 6 upregulated miRNAs (mmu-miR-21a-5p, mmu-miR-20a-5p, mmu-miR-17-5p, mmu-miR-30e-3p, mmu-miR-142a-3p, and mmu-miR-106b-3p) was confirmed by stem-loop quantitative reverse transcription PCR, which yielded results consistent with the sequencing data. These findings expand our understanding of the regulatory mechanisms of miRNAs underlying T. gondii pathogenesis and contribute new database information on cat miRNAs, opening a new perspective on the prevention and treatment of T. gondii infection.
Project description:Uterine sarcomas and mixed epithelial-mesenchymal uterine tumors are a heterogeneous group of rare tumors for which there are very few diagnostic markers available. As aberrant microRNA (miRNA) expression patterns represent putative diagnostic cancer markers, we aimed to identify miRNA expression profiles of the major uterine sarcoma subtypes and mixed epithelial-mesenchymal tumors of the uterus. Eighty-eight miRNAs were assessed by quantitative RT-PCR in cancerous and non-cancerous tissue samples collected from 29 patients with endometrial sarcoma, leiomyosarcoma, and mixed epithelial-mesenchymal tumors. Tumor and control samples significantly (P < 0.05) differed in the expression of miR-23b, miR-1, let-7f, and let-7c in endometrial sarcomas, and miR-1, let-7c, miR-133b, let-7b, miR-143, let-7a, let-7d, let-7e, let-7g, miR-222, let-7i, and miR-214 in mixed epithelial-mesenchymal tumors. All the significantly changed miRNAs were down-regulated in the malignant tissues as compared to their normal counterparts. This may suggest their tumor suppressor role in these malignancies. No statistically significant changes in miRNA expression levels were found between leiomyosarcoma tumors and controls. The identified miRNAs warrant further studies as valuable candidate markers for the differential diagnosis of uterine sarcomas from benign uterine lesions and between uterine sarcoma subtypes.
Project description:We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ? 1.5) and 19 were down-regulated (FC ? 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-?, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.