MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of other genes by transcriptional inhibition or translational repression. miR-34a is a known tumor suppressor gene and inhibits abnormal cell growth. However, its role in other tumorigenic processes is not fully known. This study aimed to investigate the action of miR-34a on cell invasion. We found that miR-34a is expressed at various levels in cervical cancer (HeLa, SiHa, C4I, C33a and CaSki) and trophoblast (BeWo and JAR) cell lines. Transient forced expression of miR-34a did not affect the proliferation of these cell lines. Computational miRNA target prediction suggested that Notch1 and Jagged1 were targets of miR-34a. By using functional assays, miR-34a was demonstrated to bind to the 3' untranslated regions of Notch1 and Jagged1. Forced expression of miR-34a altered the expression of Notch1 and Jagged1 protein as well as Notch signaling as shown by the response of Hairy Enhancer of Split-1 protein to these treatments using western blot analysis. Forced expression of miR-34a suppressed the invasiveness of HeLa and JAR cells. By using gamma-secretase inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) that interfered Notch signaling and RNA interference that knockdown Notch1 expression, we confirmed that downregulation of Notch1 reduced the invasiveness of the cells. Transfection of intracellular domain of Notch nullifies the effect of miR-34a on the invasiveness of the cells. Besides, we identified that miR-34a affected cell invasion by regulating expression of urokinase plasminogen activator through Notch. Our results provide evidence that miR-34a inhibits invasiveness through regulation of the Notch pathway and its downstream matrix degrading enzyme.

Osteosarcoma is one of the most common bone tumors. However, the genetic basis for its pathogenesis remains elusive. Here, we investigated the roles of Hedgehog (Hh) signaling in osteosarcoma development. Genetically-engineered mice with ubiquitous upregulated Hh signaling specifically in mature osteoblasts develop focal bone overgrowth, which greatly resembles the early stage of osteosarcoma. However, these mice die within three months, which prohibits further analysis of tumor progression. We therefore generated a mouse model with partial upregulated Hh signaling in mature osteoblasts and crossed it into a p53 heterozygous background to potentiate tumor development. We found that these mutant mice developed malignant osteosarcoma with high penetrance. Isolated primary tumor cells were mainly osteoblastic and highly proliferative with many characteristics of human osteosarcomas. Allograft transplantation into immunocompromised mice displayed high tumorigenic potential. More importantly, both human and mouse tumor tissues express high level of yes-associated protein 1 (Yap1), a potent oncogene that is amplified in various cancers. We show that inhibition of Hh signaling reduces Yap1 expression and knockdown of Yap1 significantly inhibits tumor progression. Moreover, long non-coding RNA H19 is aberrantly expressed and induced by upregulated Hh signaling and Yap1 overexpression. Our results demonstrate that aberrant Hh signaling in mature osteoblasts is responsible for the pathogenesis of osteoblastic osteosarcoma through Yap1 and H19 overexpression.

Minichromosome maintenance (MCM) proteins 2-7 are important in DNA replication licensing. Functional roles beyond licensing are speculated. In addition, significances in medulloblastoma (MB) remain unclear. In this study, we showed the frequent deregulation of MCM2 and MCM3 expression in 7 MB cell lines and 31 clinical samples. Moreover, DAOY and ONS76 and the clinical samples expressed elevated MCM7 transcripts with genomic gain of the gene. Immunopositivity restricted to tumor cells was found in 41, 37 and 53 out of 73 MB cases for MCM2, MCM3 and MCM7, respectively. High-MCM3 expression was associated with poor prognosis. Knockdowns of these MCMs significantly inhibited anchorage-dependent and -independent MB cell growth. The inhibition of MCM3 expression by small interfering RNA knockdown was related to G1 arrest with reduced cyclin A expression, whereas the MCM2-and MCM7-knocked-down cells arrested at G2/M with increased cyclin A expression. Interestingly, we demonstrated the links of these MCMs with cell migration and invasion using wound-healing and Transwell migration/invasion assays. Exogenous overexpression of MCM2, MCM3 and MCM7 increased anchorage-independent cell growth, and also cell migration and invasion capabilities in MB cells. The knockdown reduced the number of filopodial cells and the cells with intense stress fibers by blocking cdc42 and Rho activation. Taken together, deregulation of MCM2, MCM3 and MCM7 expression might be involved in MB tumorigenesis and we revealed undefined roles of these MCMs in control of MB cell migration and invasion.

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Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca2+ channels, and extracellular Ca2+ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca2+ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding.

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetraantennary complex-type glycans carrying Gal␤1-4GlcNAc (lacNAc) and/or GalNAc␤1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAc␣2-3(GalNAc␤1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.

The oviduct-derived embryotrophic factor, ETF-3, enhances the development of trophectoderm and the hatching process of treated embryos. Monoclonal anti-ETF-3 antibody that abolishes the embryotrophic activity of ETF-3 recognized a 115-kDa protein from the conditioned medium of immortalized human oviductal cells. Mass spectrometry analysis showed that the protein was complement C3. Western blot analysis using an antibody against C3 confirmed the cross-reactivities between anti-C3 antibody with ETF-3 and anti-ETF-3 antibody with C3 and its derivatives, C3b and iC3b. Both derivatives, but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as evidenced by the presence of C3 immunoreactivity and mRNA in the human oviduct and cultured oviductal cells. Cyclical changes in the expression of C3 immunoreactivity and mRNA were also found in the mouse oviduct with the highest expression at the estrus stage. Molecules involving in the conversion of C3b to iC3b and binding of iC3b were present in the human oviduct (factor I) and mouse preimplantation embryo (Crry and CR3), respectively. In conclusion, the present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development.Research is ongoing to optimize the embryo culture condition in human in vitro fertilization and embryo transfer programs. A large proportion of time when embryos are cultured in vitro is when they should be developing in the oviduct in vivo. Oviductal microenvironment is generally accepted to provide the best support to early embryo development. Oviductal cell coculture, the culture of embryo with oviductal cells, has been shown to improve the success rate in prospective randomized control clinical trials (1). In vitro experiments show that human oviductal cell coculture enhances the ability of the embryo at blastocyst stage to escape from its outer shell, zona pellucida, in a process known as hatching. Coculture also reduces the fragmentation rate of human embryos (2, 3) and increases the blastulation rate and total cell count per blastocyst of mouse embryos (4).Despite the success of coculture, it is not commonly used in in vitro fertilization and embryo transfer programs because of the complexity in its implementation as a service. Therefore, sequential culture, the use of different media for culturing embryos at different stages of development, is currently the method of choice for improving the outcome of the human in vitro fertilization and embryo transfer program (5). However, the culture condition in sequential culture systems is still suboptimal as the development of human embryos in these systems can be further improved by supplementation of granulocyte-macrophage colony-stimulating factor (6), a cytokine with peak expression during the preimplantation...

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of gap junction proteins, such as the connexin family members. Perturbation of their expressions may lead to spermatogenic arrest as manifested by disruption of cell-cell interaction. To explore the role(s) of connexins during spermatogenesis, we utilized the small peptide antagonistic approach to specifically deplete connexin 31, connexin 33, and pan-connexin. Three connexin peptides corresponding to the extracellular binding domain of connexin 31 and connexin 33 and to the extracellular conserved domain of connexins were designed and synthesized commercially. Peptides (at single dosage of 0.5, 1, or 2 mg per animal) were injected into rat testes and testes were collected on day 0, 1, 3, 5, 10, 15, and 30 after microinjection. In situ TUNEL assay demonstrated the induction of apoptosis in the testes after pan-connexin peptide treatment in a dose-dependent manner from day 3 and onward. Unlike the pan-connexin peptide, connexin 31 and connexin 33 peptides appeared to have little effect on inducing apoptosis and germ cell loss. CD45 staining also detected the occasional presence of infiltrating lymphocytes in the seminiferous tubules. Accompanied with the apoptotic events, two apoptotic markers, NF-kappaB and caspase 3, demonstrated a general up-regulation in their expressions. In adjacent testis sections, eliminations of connexin 31, 32, and 43 were observed. However, an induction of connexin 33 expression was detected. This suggests the versatility and functional diversity of connexins in the testis. The expression of ZO-1, the only known adaptor of connexins in the testis, was reduced and remained in a low level in the seminiferous epithelium. As such, the alterations of connexins in seminiferous epithelium may induce apoptotic signaling in the testis via the caspase 3 and the NF-kappaB pathway. This demonstrates the significant role of testicular connexins to maintain the survival of germ cells by regulating inter-cellular communications among germ cells and adjacent supporting cells during spermatogenesis. In addition, the inter-relationship between connexins and other junction proteins and associated signaling protein were investigated. After pan-connexin peptide treatment, a dys-localization of N-cadherin, an adherens junction protein, and diminution of occludin, a tight junction protein, level were detected. In addition, inductions of junction regulatory protein, cathepsin L, was observed during the course of peptide-mediated germ cell loss in the testes. In summary, pan-connexin peptide treatment triggered apoptosis and germ cell loss in the testes. This event influenced the localization and expression of different junction proteins and junction-associated protein in the testes.