Many studies have recognized that circular RNAs (circRNAs) can be promising targets for renal cell carcinoma (RCC) by acting as competing endogenous RNAs for miRNAs. This study intends to uncover the implication of a novel circRNA, circ_000926 in RCC, and how it affects tumorigenesis. Microarray-based circRNA/gene expression profiling of RCC was used to identify differentially expressed circRNAs/genes in RCC and normal tissues. miRNAs targeting the screened circRNAs/genes were predicted online, followed by analyzing circ_000926 expression in RCC. The crosstalk among circ_000926, miRNA-411 (miR-411), and CDH2 was then validated. The expression of circ_000926, miR-411, and cadherin 2 (CDH2) was up-regulated or down-regulated in RCC cells to unearth their effects on the biological behaviors of RCC cells. circ_000926 was highly expressed in RCC tissues and cell lines, whereas CDH2 was verified to be a target of miR-411. As a competing endogenous RNA, circ_000926 could directly bind to miR-411 to up-regulate CDH2. Down-regulation of circ_000926 resulted in inhibited growth, migration, and invasion abilities of RCC cells, as well as suppressed epithelial-mesenchymal transition and tumor growth. However, the inhibition of miR-411 or elevation of CDH2 reversed the antitumor effects induced by silencing circ_000926. Down-regulation of circ_000926 exerts an inhibitory effect on RCC progression through miR-411-dependent CDH2 inhibition, highlighting a potential target for RCC treatment.

Epithelial-mesenchymal transition (EMT) is a cellular development program characterized by loss of cell adhesion and increased cell mobility. It is essential for numerous processes including metastasis. In this study we have generated "aggressive" MCF-7 breast cancer cells (MCF-7-EMT), which show significantly increased invasion in contrast to wild type MCF-7 (MCF-7 WT) cells. In addition, we have analyzed, whether these cell lines differ in their metastatic behavior in vivo and in expression of invasion and/or EMT-relevant genes. Invasive behavior of different human breast cancer cell lines was tested. "Aggressive" MCF-7 cells (MCF-7-EMT) were generated using coculture and mammosphere culture techniques. To analyze whether or not MCF-7-EMT cells in contrast to MCF-7 WT cells form metastases in vivo, we assessed metastases in a nude mouse model. mRNA expression profiles of MCF-7 WT cells and MCF-7-EMT cells were compared using the Affymetrix micro array technique. Expression of selected genes was validated using real-time PCR. In addition, protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2), Vimentin (VIM), and TWIST was compared. The breast cancer cell lines showed different invasive behavior from hardly any invasion to a stronger cell movement. Coculture with osteoblast-like MG63 cells led to significantly increased cell invasion rates. The highest increase was shown using MCF-7 WT cells. Generated MCF-7-EMT cells showed significantly increased invasion as compared to MCF-7 WT cells. In 8 of 10 mice bearing orthotopically growing MCF-7-EMT tumors, we could detect metastases in liver and lung. In mice bearing MCF-7 WT tumors (n = 10), no metastases were found. MCF-7 WT cells and MCF-7-EMT cells were different in expression of 325 genes. Forty-four of the most regulated 50 invasion and/or EMT-related genes were upregulated and 6 genes were downregulated in MCF-7-EMT cells. Protein expression of mesenchymal markers CDH2, VIM, and TWIST was clearly increased in MCF-7-EMT cells. Protein expression of epithelial marker CDH1 was clearly decreased. With the breast cancer cell lines, MCF-7-EMT and MCF-7 WT cells, we have an excellent model of cells for further studies of EMT and invasion in vitro and in vivo.

Radial glial cells (RGCs) in the ventricular neuroepithelium of the dorsal telencephalon are the progenitor cells for neocortical projection neurons and astrocytes. Here we show that the adherens junction proteins afadin and CDH2 are critical for the control of cell proliferation in the dorsal telencephalon and for the formation of its normal laminar structure. Inactivation of afadin or CDH2 in the dorsal telencephalon leads to a phenotype resembling subcortical band heterotopia, also known as "double cortex," a brain malformation in which heterotopic gray matter is interposed between zones of white matter. Adherens junctions between RGCs are disrupted in the mutants, progenitor cells are widely dispersed throughout the developing neocortex, and their proliferation is dramatically increased. Major subtypes of neocortical projection neurons are generated, but their integration into cell layers is disrupted. Our findings suggest that defects in adherens junctions components in mice massively affects progenitor cell proliferation and leads to a double cortex-like phenotype.

OBJECTIVE

What is the role of miR-429 in murine embryo implantation?

CONCLUSIONS

miR-429 functions as a suppressor of epithelial-mesenchymal transition (EMT) during the process of embryo implantation by reverse regulation of Pcdh8.

BACKGROUND

MicroRNAs (miRNAs) may serve as promising regulators of embryo implantation. miR-429 was recently found to be down-regulated during embryo implantation period in a microarray analysis.

METHODS

The expression profile of miR-429 was clarified in a series of models, and the target gene was confirmed. The in vivo and in vitro effect of miR-429 on embryo implantation was examined.

METHODS

Pregnancy was produced by natural mating between female C57BL6/J mice and male mice, and a series of models, including pseudopregnancy, delayed implantation and artificial decidualization, were established. The expression profile of miR-429 during the embryo implantation period was clarified in these models. Candidate target genes of miR-429 were predicted by bioinformatic analysis and tested by luciferase activity assay. The in vivo effects of miR-429 on embryo implantation were also examined. The in vitro effects of miR-429 on EMT were studied by examining migratory and invasive capacities by transwell assay and expression profiles of cadherin family members by western blotting and qRT-PCR.

RESULTS

The expression profile of miR-429 in animal models suggested its down-regulation should be dependent on the presence and status of blastocysts and on endometrial decidualization. The luciferase activity assay showed that Pcdh8, a member of cadherin gene family, was the target gene of miR-429, and miR-429 suppressed the expression of Pcdh8 mRNA and protein. Gain-of-function of miR-429 in vivo resulted in a significant reduction of the number of implantation sites, but had little effect on fertilization. Up-regulation of miR-429 in vitro led to suppression of mesenchymal marker genes Vim, Cdh2, Zeb1 and Zeb2, and activation of epithelial marker gene Cdh1, resulting in suppression of the migratory and invasive capacities of cells. miR-429 also partially abrogated TGF-beta-induced EMT. The dysregulated expression profiles of EMT markers during embryo implantation period could be partially reversed by gain-of-function of miR-429 in vivo.

CONCLUSIONS

The association of miR-429 with other members of the miR-200 family in embryo implantation remains to be determined. The relationship between miR-429 and the cadherin family needs more intensive description and the detailed mechanism of miR-429 in regulating the cadherin family needs to be elucidated.

CONCLUSIONS

Our findings indicate that miR-429 plays a major role in embryo implantation as a suppressor of EMT by targeting Pcdh8. This information could contribute to a better understanding of the mechanisms involved in the miRNA-mediated regulation of embryo implantation, and subsequently improve treatments for infertility. The findings are consistent with that from previous research of the other members in miR-200 family in embryo implantation and in the EMT.

BACKGROUND

This study was supported by the Natural Science Foundation of China (Grant number: 81170592), and Special Fund from National Excellent Doctoral Dissertation (Grant number: 201079). There was no conflict of interest.

Metastasis and chemoresistance remain major challenges in the clinical treatment of breast cancer. Recent studies show that dysregulated microRNAs (miRNAs) play an important role in metastasis and chemoresistance development in breast cancer. Herein, we identified downregulated expression of miR-708-3p in breast cancers. In particular, miR-708-3p expression was significantly decreased in specimens from breast cancer patients with metastasis compared to that in specimens from patients with no metastasis. Consistent with clinical data, our in vitro data show that miR-708-3p was more significantly decreased in invasive breast cancer cell lines. In addition, our data show that inhibition of miR-708-3p significantly stimulated breast cancer cell metastasis and induced chemoresistance both in vitro and in vivo. In contrast, overexpression of miR-708-3p dramatically inhibited breast cancer cell metastasis and enhanced the sensitivity of breast cancer cells to chemotherapy both in vitro and in vivo. Furthermore, we identified that miR-708-3p inhibits breast cancer cell epithelial-to-mesenchymal transition (EMT) by directly targeting EMT activators, including ZEB1, CDH2 and vimentin. Taken together, our findings suggest that miR-708-3p acts as a cancer suppressor miRNA and carries out its anticancer function by inhibiting EMT in breast cancer. In addition, our findings suggest that restoration of miR-708-3p may be a novel strategy for inhibiting breast cancer metastasis and overcoming the chemoresistance of breast cancer cells.

Induced pluripotent stem cells (iPSCs) exhibit reduced efficiency and higher variability in neural differentiation compared to embryonic stem cells (ESCs). In this study, we showed that mouse iPSCs failed to efficiently give rise to neuronal cells using conventional methods previously established for driving mouse ESC differentiation. We reported a novel approach which remarkably increases neural differentiation of mouse iPSCs. This novel approach initiated embryoid body (EB) formation directly from the whole cell clones isolated from the top of feeder cells. Compared to conventional neural induction methods such as single cell suspension or monolayer culture, the cell clone-derived EB method led to a pronounced increase in directed generation of various types of neural cells including neural stem cells, motoneurons and dopaminergic neurons in response to different inducers. Through gene expression microarray analysis, we identified 14 genes that were highly expressed in the cell clone-derived EBs. Among them, we found that Cdh2, also known as N-cadherin, played important roles in controlling the neural differentiation efficiency of mouse iPSCs. Forced expression of Cdh2 in iPSCs substantially enhanced the differentiation efficiency while knocking-down of Cdh2 by shRNA blocked the neural differentiation. Our results revealed a critical role of Cdh2 in the process of efficient neural differentiation of mouse iPS cells.

The establishment of pregnancy requires bidirectional communication between the developing conceptus and the uterine endometrium. The aim of this study was to establish an in vitro coculture system with bovine trophoblast cells and uterine epithelial cells (EECs) that mimics the in vivo attachment process. We previously reported that expression of interferon tau (IFNT), a major secretory product from the trophectoderm, decreases with changes in chromatin structure when the conceptus successfully attaches to the uterine epithelium. Thus, IFNT is a good marker to assess whether attachment has successfully occurred. In this study, bovine trophoblast CT-1 cells were cultured to generate spheroids, which were then placed on type I collagen-coated plates (monoculture) or bovine EECs (coculture) with or without uterine flushings collected from Day 15 cyclic or Days 15, 17, or 19 pregnant animals. In the coculture but not the monoculture, addition of uterine flushings from Day 15 or 17 pregnant animals resulted in decreased IFNT and CDX2 mRNA expression in CT-1 spheroids, accompanied with changes in histone modifications. In monocultured CT-1 spheroids, integrin subunit ITGA8 and ITGB3 mRNAs were minimally expressed but were induced in cocultured CT-1 spheroids with or without uterine flushings. Expression of CDH2, another marker for bovine conceptus attachment to the uterine epithelium, was also induced in the cocultured CT-1 spheroids. These results suggest that this in vitro coculture system could be used to isolate processes essential for conceptus attachment to uterine EECs.

Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal (ICCs) and are the most common mesenchymal neoplasm of the gastrointestinal tract. While the majority of GISTs harbor activating mutations in either the v-kit Hardy-Zuckerman feline sarcoma viral oncogene homolog (KIT) or platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinases, approximately 10-15% of adult GISTs and 85% of pediatric GISTs lack such mutations. These "wild-type" GISTs have been reported to express high levels of the insulin-like growth factor 1 receptor (IGF1R), and IGF1R-targeted therapy of wild-type GISTs is being evaluated in clinical trials. However, it is not clear that all wild-type GISTs express IGF1R, because studies to date have predominantly focused on a particular subtype of gastric wild-type GIST that is deficient in the mitochondrial succinate dehydrogenase (SDH) complex. This study of a series of 136 GISTs, including 72 wild-type specimens, was therefore undertaken to further characterize wild-type GIST subtypes based on the relative expression of transcripts encoding IGF1R. Additional transcripts relevant to GIST biology were also evaluated, including members of the IGF-signaling pathway (IGF1, IGF2, and insulin receptor [INSR]), neural markers (CDH2[CDH: Cadherin], neurofilament, light polypeptide, LHX2 [LHX: LIM homeobox], and KIRREL3 [KIRREL: kin of IRRE like]), KIT, PDGFRA, CD34, and HIF1A. Succinate dehydrogenase complex, subunit B protein expression was also assessed as a measure of SDH complex integrity. In addition to the previously described SDH-deficient, IGF1R(high) wild-type GISTs, other SDH-intact wild-type subpopulations were defined by high relative expression of IGF1R, neural markers, IGF1 and INSR, or low IGF1R coupled with high IGF2. These results underscore the complexity and heterogeneity of wild-type GISTs that will need to be factored into molecularly-targeted therapeutic strategies.

Interaction between parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1) and low-density lipoprotein receptor-related protein 6 (Lrp6) is important for parathyroid hormone (PTH) signaling and anabolic action. Because N-cadherin has been shown to negatively regulate canonical Wnt/β-catenin signaling, we asked whether N-cadherin alters PTH signaling and stimulation of bone formation. Ablation of the N-cadherin gene (Cdh2) in primary osteogenic lineage cells resulted in increased Lrp6/PTHR1 interaction in response to PTH1-34 , associated with enhanced PTH-induced PKA signaling and PKA-dependent β-catenin C-terminus phosphorylation, which promotes β-catenin transcriptional activity. β-catenin C-terminus phosphorylation was abolished by Lrp6 knockdown. Accordingly, PTH1-34 stimulation of Tcf/Lef target genes, Lef1 and Axin2, was also significantly enhanced in Cdh2-deficient cells. This enhanced responsiveness to PTH extends to the osteo-anabolic effect of PTH, as mice with a conditional Cdh2 deletion in Osx+ cells treated with intermittent doses of PTH1-34 exhibited significantly larger gains in trabecular bone mass relative to control mice, the result of accentuated osteoblast activity. Therefore, N-cadherin modulates Lrp6/PTHR1 interaction, restraining the intensity of PTH-induced β-catenin signaling, and ultimately influencing bone formation in response to intermittent PTH administration.

Due to the fact that the treatment of breast cancer depends significantly on the molecular markers present in the cancer, including estrogen receptor (+), progesterone receptor (+) or erbB2 receptor (+), further investigation targeting triple‑negative breast cancer (TNBC) subtypes may assist in elucidating the mechanisms of recurrence of TNBC and enable the identification of novel therapeutic strategies for patients with TNBC. The aim of the present study was to compare the gene expression profiles between TNBC samples that were identified as having recurrent and non‑recurrent statuses. Between June 2011 and May 2012, a total of 30 patients with TNBC were examined using a follow-up period of at least 5 years. Their clinicopathological information was retrospectively reviewed and they were classified with a status either of recurrence [n=15 stage II (9), IIIA (2), IIIC (4)] or non‑recurrence [n=15 stage II (6), IIIA (1), IIIC (8)]. The total RNA from tissue samples obtained from the recurrent and non‑recurrent TNBC patients were used to performed oligonucleotide microarray analysis. The dataset was analyzed using GeneSpring software and validated using reverse transcription-quantitative polymerase chain reaction. Principal component analysis demonstrated that there was a marked difference in the gene expression distribution between the stage IIIc recurrent samples and early stage (stages IIa, IIb and IIIa) recurrent samples. In early stage recurrence, the significant pathway‑associated upregulated genes were matrix metalloproteinases (MMPs) and genes associated with cancer cell migration (CDH2) and cell adhesion/motility (KRAS, CDC42, RAC1, ICAM and SRGAP2). By contrast, during stage IIIc recurrence, the significant pathway‑associated upregulated genes in the recurrent samples were WNT signaling genes, including WNT 4 and WNT 16. It was concluded that there were markedly different distributions and gene expression profiles between stage IIIc recurrent TNBC tumors and early stage (IIa, IIb, IIIa) recurrent TNBC tumors, which provides important information for the development of effective treatment strategies for TNBC.

Oxidative stress induced by reactive oxygen species (ROS) overproduction would hinder bone healing process at the interface of bone/implant, yet underlying mechanism remains to be explored. To endow titanium (Ti) substrates with antioxidant activity for enhanced bone formation, multilayered structure composing of chitosan-catechol (Chi-C), gelatin (Gel) and hydroxyapatite (HA) nanofibers was constructed on Ti substrates. Surface wettability and topography of multilayer coated Ti substrates were characterized by water contact angle measurement, scanning electron microscopy and atomic force microscopy, respectively. Chi-C containing multilayer on Ti surface effectively protected osteoblasts from ROS damage, which was revealed by high level of intracellular ROS scavenging activity and reduced oxidative damage on cellular level by regulating the expression of cell adhesion related genes (integrin αv, β3, CDH11 and CDH2). Moreover, it regulated the production of cell adhesive and anti-apoptotic related proteins (p-MYPT1, p-FAK, p-Akt and Bcl-2) and pro-apoptotic critical executioners (Bax and cleaved caspase 3). Beside, the composite multilayer of Chi-C/Gel/HA nanofibers on Ti substrates promoted osteoblasts differentiation, which was evidenced by high expression levels of alkaline phosphatase activity, collagen secretion, ECM mineralization and osteogenesis-related genes expression in vitro. The in vivo experiments of μ-CT analysis, push out test and histochemistry staining further confirmed that Chi-C multilayered implant had great potential for improved early bone healing. Overall, the study offers an effective strategy for the exploration of high quality Ti implants for orthopedic applications.

OBJECTIVE

Abnormal expression of microRNA-124 (miR-124) was found in non-small cell lung cancer (NSCLC). However, the association between miR-124 and CDH2 has not been reported yet. This study aims to reveal the inhibiting effects of miR-124 on the expression of CDH2 in NSCLC.

METHODS

Quantitative real-time polymerase chain reaction was used to evaluate the expression of miR-124 and CDH2 in NSCLC tissues. Cell viability, apoptosis and invasion assays were carried out in NSCLC cell lines after transfection. The regulation mechanism was confirmed by luciferase report assay and western blot (WB).

RESULTS

Significantly decreased expression of miR-124 was found in NSCLC specimens and cell lines. Overexpression of miR-124 apparently suppressed the proliferation and invasion of NSCLC cell lines in vitro. Luciferase report assay and WB revealed that CDH2 was a target gene of miR-124. Furthermore, results of WB showed that epithelial-mesenchymal transition (EMT) could be inhibited by up-regulation of miR-124.

CONCLUSIONS

Taken together, our findings present the first evidence that miR-124 could suppress the expression of CDH2 and regulate EMT, which might lead to a potential therapeutic strategy focusing on miR-124 and CDH2 for human lung cancer.

In lung cancer, antiangiogenic strategies targeting tumor-derived endothelial cells (TECs) afford a survival advantage, but the characteristics of TECs have not been comprehensively elucidated. Herein, high-purity (> 98%) TECs were obtained, and these cells retained expression of EC markers and exhibited high viability. ITRAQ-2DLC-MS/MS was performed to profile the proteome and the heterogeneity of ECs. Only 31 of 1820 identified proteins were differentially expressed between adenocarcinoma (ADC)- and squamous cell carcinoma (SCC)-derived TECs (TEC-A and TEC-S, respectively), and cadherin-2 (CDH2) was the most significantly upregulated protein in TEC-A samples. Positive immunostaining for CDH2 (score > 3) was significantly more frequent in the endothelium of ADC tissues than in that of SCC tissues. Loss- or gain-of-function analysis showed that CDH2 significantly promoted in vitro and in vivo angiogenesis and sensitivity to the antagonist exherin. The MAPK/ERK and MAPK/JNK signaling pathways may play crucial roles in CDH2-induced HIF-1α/VEGF-mediated angiogenesis. Moreover, high CDH2 expression in TECs was significantly associated with tumor stage, visceral pleural metastasis, and decreased overall survival in patients with ADC but not SCC. Together, these data indicate the importance of CDH2 in angiogenesis and highlight its potential both for antiangiogenic therapy and as a candidate prognostic marker for ADC.

BACKGROUND

Breast cancer is one of the most frequently occurring cancers in women. In recent years, Dendrobium candidum has played a part in antihyperthyroidism and anticancer drugs. This study aims to examine the antitumor effect of D. candidum on breast cancer.

METHODS

Human breast cancer cell line MCF-7 and normal breast epithelial cell line MCF10A were used to observe the effects of D. candidum treatment on human breast cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to examine the cell proliferation of the MCF-7 and MCF10A cells. Western blot analysis and reverse transcription polymerase chain reaction were used to detect the key molecules and biomarkers in breast cancer pathology. Cell cycle was analyzed by using Becton Dickinson FACScan cytofluorometer.

RESULTS

The results indicated that D. candidum significantly decreased cell viability at different concentrations compared to the control group (P<0.05). D. candidum-treated MCF-7 cells in the G2/M phase was significantly increased compared to the control group (P<0.05). The messenger RNA levels of estrogen receptor alpha, IGFBP2, IGFBP4, and GATA3 were significantly decreased, and the messenger RNA and protein levels of ELF5, p53, p21, p18, CDH1, CDH2, and p12 were significantly increased, compared to the control group (P<0.05). The protein levels of estrogen receptor alpha, PGR, GATA3, and Ki67 were significantly decreased and the protein levels of p53 and ELF5 were significantly increased compared to the control group (P<0.05). The general apoptosis biomarker, Bcl-2, was significantly decreased and the Bax was significantly increased compared to the control group (P<0.05). In contrast to that in MCF-7, D. candidum does not affect cell proliferation at any concentration and any time points in normal breast epithelial cells, MCF10A cells.

CONCLUSIONS

D. candidum could decrease the cell viability of MCF-7 cells by inducing cell cycle arrest at the G2/M phase and regulating the key biomarkers in breast cancer cells.

DNA methylation is important in cancer development and is a promising biomarker for cancer detection. An epigenomic approach used in our previous work showed that LMX-1A is methylation-silenced in cervical cancer. LMX-1A, a LIM-homeobox gene, is known to participate in developmental events; however, there are at present no data on the role of LMX-1A in cancers. In this study, we characterized the function of this transcription factor by examining cell lines, animal models and human cervical neoplastic tissues, and found that over-expression of LMX-1A does not affect cell proliferation or the cell cycle of cervical cancer cell lines but significantly inhibits colony formation and invasion in vitro. Analysis of changes in epithelial-mesenchymal transition (EMT) markers, such as CDH1, CDH2, VIMENTIN, SNAIL, SLUG and TWIST, revealed involvement of the EMT in LMX-1A-mediated cancer invasion; this result was validated in a stable transfectant over-expressing LMX-1A with RNA interference. Xenograft studies using immunocompromised mice confirmed the suppressor effects of LMX-1A on tumour formation and distant metastasis in cervical cancer cell lines. LMX-1A immunohistochemical staining of tissue arrays containing the full spectrum of cervical neoplasms, including normal cervix, low-grade cervical intra-epithelial neoplasia (CIN), high-grade CIN, locally invasive and distant metastatic cancers, demonstrated the critical role of LMX-1A in invasion and metastasis. Furthermore, we found by analysing TGFbeta-BMP signalling that BMP4 and BMP6 are down-regulated by LMX-1A. The results of this study suggest that LMX-1A suppresses cancer invasion and metastasis in cervical cancer through an incomplete EMT.

The epididymis is responsible for posttesticular sperm maturation. Sperm maturation is dependent on the luminal microenvironments along the epididymis. Though the role of the epididymis is well established, the molecular and cellular mechanisms responsible for sperm maturation remain to be elucidated, particularly in the human, as limited biological tools exist. We have established the first stable epithelial cell lines transformed with SV40 large T antigen (LTAg) from two regions of the human adult epididymis. The cell lines are composed of homogenous populations of diploid principal cells that possess ultrastructural characteristics similar to those of human principal cells in vivo. These cells express transcripts for adherens (cadherins CDH1 and CDH2) and tight (claudins CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN8) junctions as well as desmosomes (desmoplakin, DSP). Transepithelial resistance (TER) measurements in fertile human caput epididymal cell line 1 (FHCE1) as well as the immunolocalization of tight junctional protein 1 (TJP1), occludin, and CLDN1 indicate that these cells form functional tight junctions. Furthermore, knockdown of CLDN1, CLDN3, CLDN4, or CLDN7 using specific siRNAs resulted in significant decreases in TER, suggesting that these CLDNs are essential for the barrier function of the blood-epididymis barrier. Disruption of CLDN1, CLDN3, CLDN4, and CLDN7 could, therefore, lead to epididymal dysfunction, resulting in male infertility.

The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.

Molecular mechanisms that control inner ear morphogenesis from the placode to the three-dimensional functional organ are not well understood. We hypothesize that cell-cell adhesion, mediated by cadherin molecules, contributes significantly to various stages of inner ear formation. Cadherin-2 (Cdh2) function during otic vesicle morphogenesis was investigated by examining morpholino antisense oligonucleotide knockdown and glass onion (glo) (Cdh2 mutant) zebrafish embryos. Placode formation, vesicle cavitation and specification occurred normally, but morphogenesis of the otic vesicle was affected by Cdh2 deficiency: semicircular canals were reduced or absent. Phalloidin staining of the hair cell stereocillia demonstrated that cadherin-2 (cdh2) loss-of-function did not affect hair cell number, but acetylated tubulin labeling showed that hair cell kinocilia were shorter and irregularly shaped. Statoacoustic ganglion size was significantly reduced, which suggested that neuron differentiation or maturation was affected. Furthermore, cdh2 loss-of-function did not cause a general developmental delay, since differentiation of other tissues, including eye, proceeded normally. These findings demonstrate that Cdh2 selectively affects epithelial morphogenetic cell movements, particularly semicircular canal formation, during normal ear mophogenesis.

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca(2+)/calmodulin and modulation of voltage-dependent gating by extracellular Mg(2+). Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR-376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors.

Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues.

Cell adhesion molecules, such as N-cadherin (cdh2), are essential for normal neuronal development, and as such have been implicated in an array of processes including neuronal differentiation and migration, and axon growth and fasciculation. cdh2 is expressed in neurons of the peripheral nervous system during development, but its role in these cells during this time is poorly understood. Using the transgenic zebrafish line, tg(p2xr3.2:eGFP(sl1)), we have examined the involvement of cdh2 in the formation of sensory circuits by the peripheral nervous system. The tg(p2xr3.2:eGFP(sl1)) fish allows visualization of neurons comprising the trigeminal, facial, glossopharyngeal and vagal ganglia and their axons throughout development. Reduction of cdh2 in this line was achieved by either crosses to the cdh2-mutant strain, glass onion (glo) or injection of a cdh2 morpholino (MO) into single-cell embryos. Here we show that cdh2 function is required to alter the directional vectors of growing axons upon reaching intermediate targets. The central axons enter the hindbrain appropriately but fail to turn caudally towards their final targets. Similarly, the peripheral axons extend ventrally, but fail to turn and project along a rostral/caudal axis. Furthermore, by expressing dominant negative cdh2 constructs selectively within cranial sensory ganglia (CSG) neurons, we found that cdh2 function is necessary within the axons to elicit these stereotypic turns, thus demonstrating that cdh2 acts cell autonomously. Together, our in vivo data reveal a novel role for cdh2 in the establishment of circuits by peripheral sensory neurons.

N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ∼40% of the somitic hemisegments and an ∼150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection.

BACKGROUND

Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.

RESULTS

Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1.

CONCLUSIONS

Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.