MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Recent studies indicate that miRNAs are mechanistically involved in the development of mammalian spermatogenesis. However, little work has been done to compare the miRNA expression patterns between immature and mature mouse testes. Here, we employed a miRNA microarray to detect 892 miRNAs in order to evaluate the expression patterns of miRNA. The expression of 19 miRNAs was significantly different between immature and mature individuals. Fourteen miRNAs were significantly upregulated and five miRNAs were downregulated in immature mice and this result was further confirmed by a quantitative real-time RT-PCR assay. Many target genes involved in spermatogenesis are predicted by MiRscan performing miRNA target scanning. Our data indicated specific miRNAs expression in immature mouse testis and suggested that miRNAs have a role in regulating spermatogenesis.

This research was supported by the National Natural Science Foundation of China (No. 81370693). The authors have no conflicts of interest.

Endothelial cell-specific molecules (ECSMs) play a pivotal role in the pathogenesis of many angiogenesis-related diseases. Since its initial discovery, the exact function of human ECSM2 has not been defined. In this study, by database mining, we identified a number of hypothetical proteins across species exhibiting substantial sequence homology to the human ECSM2. We showed that ECSM2 is preferentially expressed in endothelial cells and blood vessels. Their characteristic structures and unique expression patterns suggest that ECSM2 is an evolutionarily conserved gene and may have important functions. We further explored the potential roles of human ECSM2 at the molecular and cellular level. Using a reconstitution mammalian cell system, we demonstrated that ECSM2 mainly resides at the cell membrane, is critically involved in cell-shape changes and actin cytoskeletal rearrangement, and suppresses tyrosine phosphorylation signaling. More importantly, we uncovered that ECSM2 can cross-talk with epidermal growth factor receptor (EGFR) to attenuate the EGF-induced cell migration, possibly via inhibiting the Shc-Ras-ERK (MAP kinase) pathway. Given the importance of growth factor and receptor tyrosine kinase-mediated signaling and cell migration in angiogenesis-related diseases, our findings regarding the inhibitory effects of ECSM2 on EGF-mediated signaling and cell motility may have important therapeutic implications.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) with a multitude of clinical manifestations. Some CF patients develop clinically significant anemia, suggesting that CFTR may regulate hematopoiesis. Here, we report that cftr mutant zebrafish model exhibits primitive and definitive hematopoietic defects with impaired Wnt signaling. Cftr is found to interact, via its PDZ-binding domain (PDZBD), with Dishevelled (Dvl), a key component of Wnt signaling required for hematopoietic progenitor specification, thus protecting Dvl from Dapper1 (Dpr1)-induced lysosomal degradation. Defective hematopoiesis and impaired Wnt signaling in cftr mutant can be rescued by overexpression of wild-type or channel function-defective G551D mutant CFTR with an intact PDZBD, but not Cftr with mutations in the PDZBD. Analysis of human database (http://r2.amc.nl) shows that CFTR is positively correlated with DVL2 and Wnt-related hematopoietic factors in human blood system. The results reveal a previously unrecognized role of CFTR, which is independent of its channel function, in regulating DVL degradation and thus Wnt signaling required for hematopoiesis in both zebrafish and humans, providing an explanation for the anemic phenotype of CF patients.

PIWIL2, called HILI in humans, is a member of the PIWI subfamily. This subfamily has highly conserved PAZ and Piwi domains and is implicated in several critical functions, including embryonic development, stem-cell self-renewal, RNA silencing, and translational control. However, the underlying molecular mechanism remains largely unknown. Transforming growth factor-β (TGF-β) is a secreted multifunctional protein that controls several developmental processes and the pathogenesis of many diseases. TGF-β signaling is activated by phosphorylation of transmembrane serine/threonine kinase receptors, TGF-β type II (TβRII), and type I (TβRI), which are stabilized by Hsp90 via specific interactions with this molecular chaperone. Here, we present evidence that HILI suppresses TGF-β signaling by physically associating with Hsp90 in human embryonic kidney cells (HEK-293). Our research shows that HILI mediates the loss of TGF-β-induced Smad2/3 phosphorylation. We also demonstrate that HILI interacts with Hsp90 to prevent formation of Hsp90-TβR heteromeric complexes, and improves ubiquitination and degradation of TβRs dependent on the ubiquitin E3 ligase Smurf2. This work reveals a critical negative regulation level of TGF-β signaling mediated by HILI (human PIWIL2) by its ability to interact with Hsp90 and promote TβR degradation.

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Piwi proteins are required for germ cell proliferation, differentiation, and germ line stem cell maintenance. In normal tissues, human and mouse Piwil2 are primarily expressed in testis but widely expressed in tumors. However, the underlying mechanism remains largely unknown. In vertebrates, transforming growth factor (TGF)-β signaling plays an important role in patterning embryo and control of cell growth and differentiation. A previous study has shown a role for Zili, a Piwil2 gene in zebrafish, in germ cells in zebrafish. Here we report that zili functions in patterning the early embryo and inhibits TGF-β signaling. Whole mount expression analysis shows that zili expresses not only in PGCs but also in axis. Ectopic expression of zili causes fusion of the eyes and reduction of mesodermal marker genes expression, suggesting that zili functions to inhibit Nodal signaling and mesoderm formation. Genetic interaction shows that zili inhibits Nodal and bone morphogenetic protein signaling. The results of protein interaction assays identify that Zili binds to Smad4 via its N-terminal domain and prevents the formation of Smad2/3/4 and Smad1/5/9/4 complexes to antagonize TGF-β signaling. This work shows that zili plays a role in early embryogenesis beyond germ line as a novel negative regulator of TGF-β signaling, extending the function of Piwi proteins in vertebrates.

The DAZ (Deleted in AZoospermia) gene cluster on the Y chromosome is a strong candidate for the azoospermia factor. The DAZ gene was derived from an autosomal homologue, DAZL (DAZ-Like). This study was designed to assess the functional role of DAZL in human spermatogenesis. The expression patterns and mRNA transcript levels of DAZL in the testes of 17 azoospermic men were therefore examined by immunohistochemical staining and quantitative competitive reverse transcription-polymerase chain reaction. DAZL protein was expressed in the cytoplasm of primary spermatocytes and weakly in spermatogonia. It was detected in the testicular tissues of all subjects with germ cells present. The copy number of the DAZL transcript in normal spermatogenesis (n = 4), hypospermatogenesis or maturation arrest (n = 6), and Sertoli cell-only syndrome (n = 7) ranged from 1.22 x 10(6) to 1.63 x 10(6) per ng of RNA, 1.19 x 10(5) to 2.82 x 10(5) per ng of RNA and 2.83 x 10(4) to 1.23 x 10(5) per ng of RNA respectively. DAZL transcripts were lower in men with spermatogenic failure, and a significant difference was found between the three groups (P < 0.0001). This study suggests that DAZL may play an important role in the human spermatogenic processes of both mitosis and meiosis.