Browsing by Author "Winger, Quinton A., advisor"
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Item Open AccessGene regulation by let-7 microRNAs during human and sheep placental development(Colorado State University. Libraries, 2020) Ali, Asghar, author; Winger, Quinton A., advisor; Bouma, Gerrit J., advisor; Chicco, Adam, committee member; Garrity, Deborah, committee memberIntrauterine growth restriction (IUGR) is a major cause of perinatal morbidity and mortality and affects more than 30 million infants every year across the world. Its occurrence is 5–15% of all pregnancies in the United States and 10-55% in developing countries. Most common etiology of IUGR is impaired placental development. Structural and functional abnormalities in placenta can also lead to preeclampsia (PE), still birth and spontaneous abortion. Conditions like IUGR and PE are usually not detected until later stages of gestation. Hence, there is a need to better understand the placental development and function to improve diagnosis and treatment of placenta-associated disorders. In this study, we investigated genetic pathways regulated by let-7 miRNAs and their potential role in pathogenesis of malformed placenta. Let-7 miRNAs are markers of cell differentiation and reduce the expression of several genes by translational repression. Biogenesis of let-7 miRNAs is suppressed by LIN28 which is an RNA binding oncofetal protein with two paralogs, LIN28A and LIN28B. LIN28 is high and let-7 miRNAs are low in proliferating stem cells whereas LIN28 is low and let-7 miRNAs are high in differentiated cells. LIN28-let-7 axis determines fate of cells by regulating the expression of genes associated with cell proliferation and differentiation. In human placenta, LIN28 is mainly found in trophoblast cells and the fetal portion of placenta comprises mainly of trophoblast cells. We found that in term human placentas from IUGR pregnancies, LIN28 is low and let-7 miRNAs are high compared to placentas from control placentas. We further saw a reduction in the expression of AT-Rich Interaction Domain 3A (ARID3A) and AT-Rich Interaction Domain 3B (ARID3B). ARID3A and ARID3B promote cell proliferation by transcriptional regulation of stemness genes. In immortalized first trimester human trophoblast (ACH-3P) cells, ARID3A and ARID3B complex with lysine demethylase 4C (KDM4C) to make the ARID3B-complex. This complex binds the promoter regions of proliferation-associated genes such as high mobility group AT-hook 1 (HMGA1), transcriptional regulator Myc-like (c-MYC), vascular endothelial growth factor A (VEGF-A) and Wnt family member 1 (WNT1). These genes are also targeted by let-7 miRNAs. LIN28 knockout ACH-3P cells have significantly increased let-7 miRNAs and significantly reduced HMGA1, c-MYC, VEGF-A and WNT1. We also saw significant reduction in ARID3A and ARID3B in LIN28 knockout ACH-3P cells. ACH-3P cells with ARID3B knockout also showed significant reduction in HMGA1, c-MYC, VEGF-A and WNT1. Both LIN28 knockout and ARID3B knockout in ACH-3P cells resulted in reduced cell proliferation compared to control. These results suggest that proliferation-associated genes in trophoblast cells are regulated through LIN28-let-7-ARID3B pathway. Trophectoderm (TE) specific knockdown of LIN28 in sheep led to reduced conceptus elongation at day 16 of gestation. Furthermore, LIN28 knockdown day 16 TE had significantly increased let-7 miRNAs and significantly reduced expression of proliferation factors including insulin like growth factor 2 mRNA binding proteins (IGF2BPs), HMGA1, ARID3B and c-MYC. From these findings, we interpret that LIN28-let-7-ARID3B pathways regulates proliferation of trophoblast cells and is potentially associated with etiology of IUGR. Item Open AccessImpact of chorionic somatomammotropin in vivo RNA interference phenotype on uteroplacental expression of the IGF axis(Colorado State University. Libraries, 2023) Hord, Taylor, author; Anthony, Russel V., advisor; Winger, Quinton A., advisor; Bruemmer, Jason, committee member; Kading, Rebekah, committee memberWhile fetal growth is dependent on many factors, optimal placental function is a prerequisite for a normal pregnancy outcome. The majority of fetal growth restricted (FGR) pregnancies result from placental insufficiency (PI). The insulin-like growth factors (IGF1 and IGF2) not only stimulate fetal growth, but also placental development and function. Previously, we demonstrated that in vivo RNA interference (RNAi) of the placental hormone, chorionic somatomammotropin (CSH), resulted in two phenotypes. One phenotype exhibits significant placental and fetal growth restriction (PI-FGR), impaired placental nutrient transport, and significant reductions in umbilical insulin and IGF1. The other phenotype does not exhibit statistically significant changes in placental or fetal growth (non-FGR). It was our objective to further characterize these two phenotypes by determining the impact of CSH RNAi on placental (maternal caruncle and fetal cotyledon) expression of the IGF axis. The trophectoderm of hatched blastocysts (9 days of gestation, dGA) were infected with a lentivirus expressing either a non-targeting sequence (NTS RNAi) control or CSH-specific shRNA (CSH RNAi) prior to embryo transfer into synchronized recipient ewes. At ≈125 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies. Nutrient uptakes were determined and tissues were harvested at necropsy. In both CSH RNAi non-FGR and PI-FGR pregnancies, uterine blood flow was significantly reduced (P≤0.05), while umbilical blood flow (P≤0.01), both uterine and umbilical glucose and oxygen uptakes (P≤0.05), and umbilical concentrations of insulin and IGF1 (P≤0.05) were reduced in CSH RNAi PI-FGR pregnancies. Fetal cotyledon IGF1 mRNA concentration was reduced (P≤0.05) in CSH RNAi PI-FGR pregnancies, whereas neither IGF1 nor IGF2 mRNA concentrations were impacted in the maternal caruncles, and either placental tissue in the non-FGR pregnancies. Fetal cotyledon IGF1R and IGF2R mRNA concentrations were not impacted for either phenotype, yet IGF2R was increased (P≤0.01) in the maternal caruncles of CSH RNAi PI-FGR pregnancies. For the IGF binding proteins (IGFBP1, IGFBP2, IGFBP3), only IGFBP2 mRNA concentrations were impacted, with elevated IGFBP2 mRNA in both the fetal cotyledon (P≤0.01) and maternal caruncle (P=0.08) of CSH RNAi non-FGR pregnancies. These data support the importance of IGF1 in placental growth and function, but may also implicate IGFBP2 in salvaging placental growth in non-FGR pregnancies. Item Open AccessPluripotency factors and miRNAs in human ovarian cancer cells and their secreted exosomes regulate gene expression and phenotype(Colorado State University. Libraries, 2014) Enriquez, Vanessa Ann, author; Bouma, Gerrit J., advisor; Winger, Quinton A., advisor; Bailey, Susan M., committee member; Duval, Dawn L., committee memberOvarian cancer is the fifth most deadly cancer among women in the United States and the most lethal gynecological malignancy in the world. Recent studies reveal that human tumor cells release cell-secreted vesicles called exosomes. Exosomes are endosome-derived vesicles containing bioactive materials, including RNAs and miRNAs that can be detected in body fluids. Importantly, pluripotency factor LIN28, a regulator of let-7 miRNAs, is present in ovarian cancer cells. High LIN28A and low let-7 miRNA levels are associated with aggressive IGROV1 human ovarian cancer cells (Piskounova et al., 2011) so we compared this to low LIN28A and high let-7 miRNA levels in less aggressive OV420 human ovarian cancer cells. Moreover, let-7b, let-7c, let-7g, and let-7i miRNA signatures were also present in their secreted exosomes. We hypothesized that ovarian cancer cell-secreted exosomes are taken up by target cells and induce changes in gene expression and cell behavior. Our data revealed that IGROV1 secreted exosomes taken up by HEK293 cells lead to significantly higher LIN28A mRNA levels, but not LIN28A protein levels and did not significantly change LIN28B mRNA or protein levels. However, IGROV1 exosome exposure to HEK293 cells did significantly increase invasion and migration. In addition, various genes involved in epithelial-mesenchymal transition, including TIMP1 (25-fold higher), NOTCH1 (11-fold-higher), SNAI1 (SNAIL) (7-fold higher), CDH1 (6-fold higher), (MMP9 (4-fold higher), and ZEB1 (3-fold higher), were significantly higher in HEK293 cells following uptake of IGROV1 secreted exosomes. We also postulated that ovarian cancer cell-secreted exosomes contain a distinct RNA signature capable of inducing phenotypic changes in target cells, as well as distinguishing aggressive, advanced ovarian exosomes from less aggressive ovarian exosomes. Since IGROV1 cells are a more aggressive epithelial ovarian cancer cell line, while OV420 cells are a less aggressive cell line, we performed RNAseq transcriptome analysis on exosomes from IGROV1 cells and exosomes from OV420 cells. The results yielded 312 differentially expressed RNAs. Future studies will allow us to detect the RNAs present in exosomes from urine in individuals with early stage versus late stage ovarian cancer. This may enable investigators to distinguish a poor prognosis group from a good prognosis group leading to a potential biomarker for ovarian cancer detection. Data from this study is important for elucidating the role ovarian cancer cell-secreted exosomes have on early metastasis and tumor progression, an area in ovarian cancer biology in critical need of advancement. Item Open AccessRegulation of trophoblast stem cell maintenance and differentiation by LIN28 and AP-2γ(Colorado State University. Libraries, 2010) Fromme, Brittany A., author; Winger, Quinton A., advisor; Bouma, Gerrit J., advisor; Bailey, Susan M., committee member; Anthony, Russell V., committee memberThe placenta is a unique organ essential for survival of the fetus in all eutherian mammals. Failure to develop a normal placenta in humans can lead to diseases, such as pre-eclampsia, with high morbidity and mortality for both the mother and the fetus. These diseases are thought to be caused by abnormal proliferation and differentiation of cells in the placenta. A mouse trophoblast stem (TS) cell culture system is a useful tool in studying TS cell proliferation and differentiation into trophoblast giant cells (TGCs). TS cells cultured in proliferative media (70% conditioned media, 30% TS media, FGF4, and heparin sulfate) will remain proliferative, and TS cells cultured under differentiation media (100% TS media) will differentiate into TGCs. LIN28 is a protein that regulates mRNAs and miRNAs, and is abundantly expressed in many undifferentiated tissues. AP- 2y has been shown to be essential for TS cell maintenance and TGC formation. AP-2y null mutants display embryonic lethality at E7.5 due to a severely disrupted extraembryonic portion of the embryo. In TS cells, AP-2y has been shown to bind to the promoter region of Lin28. This study investigates the hypothesis that Liti28 and Ap-2y are necessary regulators of trophoblast stem cell maintenance and differentiation into TGCs. This study shows the pluripotency genes, Lin28, Sox2, and NrObl, to be differentially expressed in proliferating TS cells and differentiated TGCs. MiRNAs can be used as markers for proliferation or differentiation. 28 significantly different miRNAs were detected between TS cells and TGCs, 18 up-regulated in TGCs and 9 downregulated in TGCs. Expression of the miR-290 family, initially thought to be ES cell specific, was detected in proliferating TS cells suggesting TS cells have similar miRNA mediated regulation of proliferation compared to ES cells. The Let-7 family of miRNAs was found to be up-regulated in differentiated TGCs. The Let-7 family has been shown to be regulated by LIN28, where LIN28 prevents accumulation of mature Let-7 miRNAs. In this study Lin28 was highly expressed in proliferating cells and the Let-7’s are upregulated in differentiated TGCs. Lin28 function in TS cells was assessed by knocking down Lin28 using shRNA lentiviral technology. Lin28 knockdown TS cells were used to observe results of knockdown. We obtained a 78% reduction of Lin28 mRNA, but found that loss of Lin28 in TS cells did not affect morphology, proliferation or differentiation. AP-2y null TS cells grown in culture fail to differentiate morphologically into TGCs. Lin28, Sox2, and NrObl show no difference in expression when grown in conditions to differentiate the cells, indicating a failure of AP-2y null TS cells to differentiate into TGCs. RO3306 is a compound used to block Cyclin-dependent Protein Kinase 1 and force endoreduplication, causing TS cells to differentiate into TGCs. AP-2y null TS cells cannot be forced to differentiate into TGCs, and instead undergo cell death, when cultured with RO3306. Additionally, AP-2y null TS cells express the pluripotency markers Oct4, Stella, and Nanog which only are expressed in ES cells and germ cells. MiRNA profiling of AP-2y null TS cells indicates that cells in proliferative conditions resemble wild type counterparts, but when proliferative conditions are removed we observe an increase in expression of the ES cell specific miR-302 cluster. While there was no effect of proliferation in wild type cells, loss of Lin28 in AP-2y null TS cells via lentiviral knockdown leads to a partial rescue of TGC formation. This suggests that Liii28 must be down-regulated in order for TGC formation, and that AP-2y regulates Lin28 in TS cells. Taken together these data suggest a role for Lin28 in mouse TS cell proliferation and differentiation, where Lin28 must become down regulated in order for differentiation into TGCs. AP-2y has been shown to bind to the Lin28 promoter in TS cells; this regulation enables TS cell differentiation into TGCs. This study also shows the necessity of AP-2y for TS cell differentiation into TGCs; loss of AP-2y leads to a more pluripotent state rather than allowing for differentiation. Loss of AP-2y leads to expression of pluripotency markers Oct4, Nanog, and Stella, and the ES cell specific miR-302 cluster, indicating an increase in pluripotency. We conclude that AP-2y and LIN28 are essential molecular regulators of TS cell proliferation and differentiation. Item Open AccessTcfap2c regulation of primordial germ cell development(Colorado State University. Libraries, 2011) Guttormsen, Jillian Bosick, author; Winger, Quinton A., advisor; Bouma, Gerrit J., committee member; Anthony, Russell V., committee member; Garrity, Deborah, committee memberThe development of germ cells during embryonic development is driven by a complex expression pattern of genes. The transcription factor Tcfap2c is expressed in germ cells throughout development from specification to adult sperm and oocytes. Tcfap2c expression is first seen in primordial germ cells around embryonic day (E)6.75 and has been classified as a germ cell specification gene. This study implicates Tcfap2c as a potential key factor in germ cells during specification, proliferation, migration and differentiation. In order to investigate the role of Tcfap2c in germ cells, we utilized the Cre/loxP conditional gene mutation strategy. Cre/loxP allows us to overcome the early embryonic lethality that arises from loss of Tcfap2c in traditional knock-out mice by creating Tcfap2c null mutation in specifically-targeted tissues. We created Tcfap2c mutant mice using the epiblast-specific Sox2-Cre model. Mutant ovaries from this model failed to express both germ cell specific markers and meiotic markers at E12.5. Immunohistochemistry at E18.5 failed to detect the germ cell specific marker NOBOX or the meiotic protein SYCP3, which confirmed that Sox2-Cre, Tcfap2c mutant mice lacked germ cells at late embryonic stages. However, Sox2-Cre, Tcfap2c mutant mice die prior to or at birth preventing us from studying adult gonads from these mice. To this end we used tamoxifen inducible ERTM-Cre mice to create Tcfap2c mutation in adult animals. We assessed ERTM-Cre, Tcfap2c mutant animals for fertility and gametogenesis; surprisingly, fertility, spermatogenesis and oogenesis were not affected in Tcfap2c mutant gonads. These results show that Tcfap2c is not necessary for adult maturation of gonocytes to produce mature sperm and oocytes. However, Sox2-Cre, Tcfap2c mutants lack germ cells indicating that Tcfap2c is necessary during fetal germ cell differentiation. The Sox2-Cre model was limited because Tcfap2c was deleted in the entire embryo and the mutants died at birth. Prdm1-Cre was used to produce a mouse where Tcfap2c is only deleted in germ cells beginning around specification. Prdm1-Cre, Tcfap2c mutants initially specified germ cell-like cells at E7.5; however, by E8.5 the germ cell numbers were decreased and they had not initiated migration towards the genital ridges. By E9.5 few if any germ cells were observed in Prdm1-Cre, Tcfap2c mutants. At E12.5 no germ cells were seen in Prdm1-Cre, Tcfap2c mutant XX or XY gonads. Adult ovaries and testes from Prdm1-Cre, Tcfap2c mutant mice were noticeably smaller than littermate controls and showed no oogenesis or spermatogenesis. The Prdm1-Cre model showed that mutation of Tcfap2c results in loss of germ cells in embryos by E9.5 suggesting that Tcfap2c plays a role during germ cell specification, proliferation and migration. We identified Tcfap2c as an important factor during early germ cell development; however, Tcfap2c expression is observed in germ cells well past specification. We believe that Tcfap2c is present in germ cells during during fetal gonad differentiation because it plays a role in regulating the gene expression pathways necessary for this event. We show that Tcfap2c is expressed in germ cells during the period of fetal gonad differentiation. Gene expression analysis of gonads from E11.5-13.5 reveals Tcfap2c as the most highly expressed member of the Tcfap2 family member. Tcfap2c is a member of a transcription factor family that regulates gene expression by binding consensus sequences within target gene promoters. TCFAP2 binding sites are present in promoter regions of germ cell specific genes Cadherin1 (Cdh1) and Kit oncogene (Kit), as well as in the promoter regions of genes involved in regulating pluripotency High mobility group AT-hook 2 (Hmga2), Nanog homeobox (Nanog) and Lin28. Using chromatin immunoprecipitation we demonstrate that TCFAP2C binds the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28. The interaction between TCFAP2C and the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28 indicates that Tcfap2c likely plays a functional role in the regulation of these genes. These genes are necessary for germ cell survival, migration and pluripotency. In conclusion, our results provide a new understanding of the role of Tcfap2c during different stages of germ cell development from specification to differentiation. Item Open AccessThe role of LIN28 in the molecular regulation of placenta development and function(Colorado State University. Libraries, 2013) Seabrook, Jill L., author; Winger, Quinton A., advisor; Clay, Colin M., committee member; Bouma, Gerrit J., committee member; DeLuca, Jennifer G., committee memberTo view the abstract, please see the full text of the document.