Pseudouridine synthase 1 promotes hepatocellular carcinoma through mRNA pseudouridylation to enhance the translation of oncogenic mRNAs.

BACKGROUND AND AIMS: Pseudouridine is a prevalent RNA modification and is highly present in the serum and urine of patients with HCC. However, the role of pseudouridylation and its modifiers in HCC remains unknown. We investigated the function and underlying mechanism of pseudouridine synthase 1 (PUS1) in HCC. APPROACH AND RESULTS: By analyzing the TCGA data set, PUS1 was found to be significantly upregulated in human HCC specimens and positively correlated with tumor grade and poor prognosis of HCC. Knockdown of PUS1 inhibited cell proliferation and the growth of tumors in a subcutaneous xenograft mouse model. Accordingly, increased cell proliferation and tumor growth were observed in PUS1-overexpressing cells. Furthermore, overexpression of PUS1 significantly accelerates tumor formation in a mouse HCC model established by hydrodynamic tail vein injection, while knockout of PUS1 decreases it. Additionally, PUS1 catalytic activity is required for HCC tumorigenesis. Mechanistically, we profiled the mRNA targets of PUS1 by utilizing surveying targets by apolipoprotein B mRNA-editing enzyme 1 (APOBEC1)-mediated profiling and found that PUS1 incorporated pseudouridine into mRNAs of a set of oncogenes, thereby endowing them with greater translation capacity. CONCLUSIONS: Our study highlights the critical role of PUS1 and pseudouridylation in HCC development, and provides new insight that PUS1 enhances the protein levels of a set of oncogenes, including insulin receptor substrate 1 (IRS1) and c-MYC, by means of pseudouridylation-mediated mRNA translation.

Epstein-Barr virus-derived circular RNA LMP2A induces stemness in EBV-associated gastric cancer.

Cancer stem cells (CSCs) are cancer-initiating cells that are not only a source of tumorigenesis but also the cause of tumour progression, metastasis and therapy resistance. EBV-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer with unique clinicopathological and molecular features. However, whether CSCs exist in EBVaGC, and the tumorigenic mechanism of EBV, remains unclear. Here, NOD/SCID mice were injected subcutaneously with the EBVaGC cell line SNU719 and treated with 5-fluorouracil weekly. Successive generations of xenografts yielded a highly malignant EBVaGC cell line, SNU-4th, which displays properties of CSCs and mainly consists of CD44+ CD24- cells. In SNU-4th cells, an EBV-encoded circRNA, ebv-circLMP2A, expression increased and plays crucial roles in inducing and maintaining stemness phenotypes through targeting miR-3908/TRIM59/p53 axis. Additionally, high expression of ebv-circLMP2A is significantly associated with metastasis and poor prognosis in patients with EBVaGC. These findings not only provide evidence for the existence of CSCs in EBVaGC and elucidate the pathogenic mechanism of ebv-circLMP2A in EBVaGC, but also provide a promising therapeutic target for EBVaGC.

N6-methyladenine demethylase ALKBH1 inhibits the differentiation of skeletal muscle.

DNA N6-methyladenine (N6-mA) was recently recognized as a new epigenetic modification in mammalian genome, and ALKBH1 was discovered as its demethylase. Knock-out mice studies revealed that ALKBH1 was indispensable for normal embryonic development. However, the function of ALKBH1 in myogenesis is largely unknown. In this study, we found that N6-mA showed a steady increase, going along with a strong decrease of ALKBH1 during skeletal muscle development. Our results also showed that ALKBH1 enhanced proliferation and inhibited differentiation of C2C12 cells. Genome-wide transcriptome analysis and reporter assays further revealed that ALKBH1 accomplished the differentiation inhibiting function by regulating a core set of genes and multiple signaling pathways, including increasing chemokine (C-X-C motif) ligand 14 (CXCL14) and activating ERK signaling. Taken together, our results demonstrated that ALKBH1 is critical for the myogenic differentiation of C2C12 cells, and suggested that N6-mA might be a new epigenetic mechanism for the regulation of myogenesis.

The landscape of long noncoding RNA-involved and tumor-specific fusions across various cancers.

The majority of the human genome encodes long noncoding RNA (lncRNA) genes, critical regulators of various cellular processes, which largely outnumber protein-coding genes. However, lncRNA-involved fusions have not been surveyed and characterized yet. Here, we present a systematic study of the lncRNA fusion landscape across cancer types and identify >30 000 high-confidence tumor-specific lncRNA fusions (using 8284 tumor and 6946 normal samples). Fusions positively correlated with DNA damage and cancer stemness and were specifically low in microsatellite instable (MSI)-High or virus-infected tumors. Moreover, fusions distribute differently among cancer molecular subtypes, but with shared enrichment in tumors that are microsatellite stable (MSS), with high somatic copy number alterations (SCNA), and with poor survival. Importantly, we find a potentially new mechanism, mediated by enhancer RNAs (eRNA), which generates secondary fusions that form densely connected fusion networks with many fusion hubs targeted by FDA-approved drugs. Finally, we experimentally validate functions of two tumor-promoting chimeric proteins derived from mRNA-lncRNA fusions, KDM4B-G039927 and EPS15L1-lncOR7C2-1. The EPS15L1 fusion protein may regulate (Gasdermin E) GSDME, critical in pyroptosis and anti-tumor immunity. Our study completes the fusion landscape in cancers, sheds light on fusion mechanisms, and enriches lncRNA functions in tumorigenesis and cancer progression.

METTL3 regulates skeletal muscle specific miRNAs at both transcriptional and post-transcriptional levels.

METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations.

BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response.

The endoplasmic reticulum (ER) is classically linked to metabolic homeostasis via the activation of unfolded protein response (UPR), which is instructed by multiple transcriptional regulatory cascades. BRCA1 associated protein 1 (BAP1) is a tumor suppressor with de-ubiquitinating enzyme activity and has been implicated in chromatin regulation of gene expression. Here we show that BAP1 inhibits cell death induced by unresolved metabolic stress. This prosurvival role of BAP1 depends on its de-ubiquitinating activity and correlates with its ability to dampen the metabolic stress-induced UPR transcriptional network. BAP1 inhibits glucose deprivation-induced reactive oxygen species and ATP depletion, two cellular events contributing to the ER stress-induced cell death. In line with this, Bap1 KO mice are more sensitive to tunicamycin-induced renal damage. Mechanically, we show that BAP1 represses metabolic stress-induced UPR and cell death through activating transcription factor 3 (ATF3) and C/EBP homologous protein (CHOP), and reveal that BAP1 binds to ATF3 and CHOP promoters and inhibits their transcription. Taken together, our results establish a previously unappreciated role of BAP1 in modulating the cellular adaptability to metabolic stress and uncover a pivotal function of BAP1 in the regulation of the ER stress gene-regulatory network. Our study may also provide new conceptual framework for further understanding BAP1 function in cancer.

Long non-coding RNAs in cancer metabolism.

Altered cellular metabolism is an emerging hallmark of cancer. Accumulating recent evidence links long non-coding RNAs (lncRNAs), a still poorly understood class of non-coding RNAs, to cancer metabolism. Here we review the emerging findings on the functions of lncRNAs in cancer metabolism, with particular emphasis on how lncRNAs regulate glucose and glutamine metabolism in cancer cells, discuss how lncRNAs regulate various aspects of cancer metabolism through their cross-talk with other macromolecules, explore the mechanistic conceptual framework of lncRNAs in reprogramming metabolism in cancers, and highlight the challenges in this field. A more in-depth understanding of lncRNAs in cancer metabolism may enable the development of novel and effective therapeutic strategies targeting cancer metabolism.

Conservation and divergence of transcriptional coregulations between box C/D snoRNA and ribosomal protein genes in Ascomycota.

Coordinated assembly of the ribosome is essential for proper translational activity in eukaryotic cells. It is therefore critical to coordinate the expression of components of ribosomal programs with the cell's nutritional status. However, coordinating expression of these components is poorly understood. Here, by combining experimental and computational approaches, we systematically identified box C/D snoRNAs in four fission yeasts and found that the expression of box C/D snoRNA and ribosomal protein (RP) genes were orchestrated by a common Homol-D box, thereby ensuring a constant balance of these two genetic components. Interestingly, such transcriptional coregulations could be observed in most Ascomycota species and were mediated by different cis-regulatory elements. Via the reservation of cis elements, changes in spatial configuration, the substitution of cis elements, and gain or loss of cis elements, the regulatory networks of box C/D snoRNAs evolved to correspond with those of the RP genes, maintaining transcriptional coregulation between box C/D snoRNAs and RP genes. Our results indicate that coregulation via common cis elements is an important mechanism to coordinate expression of the RP and snoRNA genes, which ensures a constant balance of these two components.

Deciphering the transcriptional regulation of microRNA genes in humans with ACTLocater.

Understanding the transcriptional regulation of microRNAs (miRNAs) is extremely important for determining the specific roles they play in signaling cascades. However, precise identification of transcription factor binding sites (TFBSs) orchestrating the expressions of miRNAs remains a challenge. By combining accessible chromatin sequences of 12 cell types released by the ENCODE Project, we found that a significant fraction (~80%) of such integrated sequences, evolutionary conserved and in regions upstream of human miRNA genes that are independently transcribed, were preserved across cell types. Accordingly, we developed a computational method, Accessible and Conserved TFBSs Locater (ACTLocater), incorporating this chromatin feature and evolutionary conservation to identify the TFBSs associated with human miRNA genes. ACTLocater achieved high positive predictive values, as revealed by the experimental validation of FOXA1 predictions and by the comparison of its predictions of some other transcription factors (TFs) to empirical ChIP-seq data. Most notably, ACTLocater was widely applicable as indicated by the successful prediction of TF → miRNA interactions in cell types whose chromatin accessibility profiles were not incorporated. By applying ACTLocater to TFs with characterized binding specificities, we compiled a novel repository of putative TF → miRNA interactions and displayed it in ACTViewer, providing a promising foundation for future investigations to elucidate the regulatory mechanisms of miRNA transcription in humans.

RNA-binding protein CCDC137 activates AKT signaling and promotes hepatocellular carcinoma through a novel non-canonical role of DGCR8 in mRNA localization.

BACKGROUND: RNA binding proteins (RBPs)-regulated gene expression play a vital role in various pathological processes, including the progression of cancer. However, the role of RBP in hepatocellular carcinoma (HCC) remains much unknown. In this study, we aimed to explore the contribution of RBP CCDC137 in HCC development. METHODS: We analyzed the altered expression level and clinical significance of CCDC137 in database and HCC specimens. In vitro cell assays and in vivo spontaneous mouse models were used to assess the function of CCDC137. Finally, the molecular mechanisms of how CCDC137 regulates gene expression and promotes HCC was explored. RESULTS: CCDC137 is aberrantly upregulated in HCC and correlates with poor clinical outcomes in HCC patients. CCDC137 markedly promoted HCC proliferation and progression in vitro and in vivo. Mechanistically, CCDC137 binds with FOXM1, JTV1, LASP1 and FLOT2 mRNAs, which was revealed by APOBEC1-mediated profiling, to increase their cytoplasmic localization and thus enhance their protein expressions. Upregulation of FOXM1, JTV1, LASP1 and FLOT2 subsequently synergistically activate AKT signaling and promote HCC. Interestingly, we found that CCDC137 binds with the microprocessor protein DGCR8 and DGCR8 has a novel non-canonical function in mRNA subcellular localization, which mediates the cytoplasmic distribution of mRNAs regulated by CCDC137. CONCLUSIONS: Our results identify a critical proliferation-related role of CCDC137 and reveal a novel CCDC137/DGCR8/mRNA localization/AKT axis in HCC progression, which provide a potential target for HCC therapy.

The Conserved LncRNA DIO3OS Restricts Hepatocellular Carcinoma Stemness by Interfering with NONO-Mediated Nuclear Export of ZEB1 mRNA.

Hepatocellular carcinoma (HCC) is an aggressive and fatal disease caused by a subset of cancer stem cells (CSCs). It is estimated that there are approximately 100 000 long noncoding RNAs (lncRNAs) in humans. However, the mechanisms by which lncRNAs affect tumor stemness remain poorly understood. In the present study, it is found that DIO3OS is a conserved lncRNA that is generally downregulated in multiple cancers, including HCC, and its low expression correlates with poor clinical outcomes in HCC. In in vitro cancer cell lines and an in vivo spontaneous HCC mouse model, DIO3OS markedly represses tumor development via its suppressive role in CSCs through downregulation of zinc finger E-box binding homeobox 1 (ZEB1). Interestingly, DIO3OS represses ZEB1 post-transcriptionally without affecting its mRNA levels. Subsequent experiments show that DIO3OS interacts with the NONO protein and restricts NONO-mediated nuclear export of ZEB1 mRNA. Overall, these findings demonstrate that the DIO3OS-NONO-ZEB1 axis restricts HCC development and offers a valuable candidate for CSC-targeted therapeutics for HCC.

Liver-enriched transcription factors regulate microRNA-122 that targets CUTL1 during liver development.

UNLABELLED: MicroRNA-122 (miR-122) is a liver-specific microRNA whose expression is specifically turned on in the mouse liver during embryogenesis, thus it is expected to be involved in liver development. However, the role of miR-122 in liver development and its potential underlying mechanism remain unclear. Here, we show that the expression of miR-122 is closely correlated with four liver-enriched transcription factors (LETFs)-hepatocyte nuclear factor (HNF) 1α, HNF3ß, HNF4α, and CCAAT/enhancer-binding protein (C/EBP) α-in the livers of developing mouse embryos and in human hepatocellular carcinoma (HCC) cell lines. Correspondingly, promoter analysis revealed that these LETFs are coordinately involved in the transcriptional regulation of miR-122, and three HNFs directly bind to the miR-122 promoter as transcriptional activators. Using a luciferase reporter system, we identified a group of miR-122 targets involved in proliferation and differentiation regulation. Among these targets, the most prominently repressed target was CUTL1, a transcriptional repressor of genes specifying terminal differentiation in multiple cell lineages, including hepatocytes. We show that CUTL1 expression is gradually silenced at the posttranscriptional level during mouse liver development. Overexpression and knockdown studies both showed that miR-122 repressed CUTL1 protein expression in HCC cell lines. Finally, we show that the stable restoration of miR-122 in HepG2 cells suppresses cellular proliferation and activates the expression of three hepatocyte functional genes, including the cholesterol-7α hydroxylase gene (CYP7A1), a known target of CUTL1 in hepatocytes. CONCLUSION: Our study provides a model in which miR-122 functions as an effector of LETFs and contributes to liver development by regulating the balance between proliferation and differentiation of hepatocytes, at least by targeting CUTL1.

Human DUBs' gene expression and regulation in antiviral signaling in response to poly (I:C) treatment.

Type I interferons (IFNs) play a central role in host defense against viral infection. Multiple posttranslational modifications including ubiquitination and deubiquitination regulate the function of diverse molecules in type I IFN signaling. Many ubiquitin ligase enzymes, such as those of the TRAF and TRIM families, have been shown to participate in the production of type I IFNs and inflammatory cytokines. However, the function of deubiquitinating enzymes (DUBs), a protein family that counteracts the action of protein ubiquitination, on the regulation of antiviral immune responses is not well understood. In this study, we used the broad-spectrum DUB inhibitor G5 to reveal their function in antiviral signaling, and then systematically analyzed mRNA expression of the DUB genes upon poly (I:C) treatment in THP-1 cells. Based on this analysis, we cloned some DUB genes whose expression changed and determined their function in antiviral signaling. Taken together, we present a comprehensive DUB gene expression analysis in THP-1 cells, and suggest the involvement of this family of proteins in the regulation of host antiviral activities.

Dynamic m6A mRNA Methylation Reveals the Role of METTL3/14-m6A-MNK2-ERK Signaling Axis in Skeletal Muscle Differentiation and Regeneration.

N6-methyladenosine (m6A) RNA methylation has emerged as an important factor in various biological processes by regulating gene expression. However, the dynamic profile, function and underlying molecular mechanism of m6A modification during skeletal myogenesis remain elusive. Here, we report that members of the m6A core methyltransferase complex, METTL3 and METTL14, are downregulated during skeletal muscle development. Overexpression of either METTL3 or METTL14 dramatically blocks myotubes formation. Correspondingly, knockdown of METTL3 or METTL14 accelerates the differentiation of skeletal muscle cells. Genome-wide transcriptome analysis suggests ERK/MAPK is the downstream signaling pathway that is regulated to the greatest extent by METTL3/METTL14. Indeed, METTL3/METTL14 expression facilitates ERK/MAPK signaling. Via MeRIP-seq, we found that MNK2, a critical regulator of ERK/MAPK signaling, is m6A modified and is a direct target of METTL3/METTL14. We further revealed that YTHDF1 is a potential reader of m6A on MNK2, regulating MNK2 protein levels without affecting mRNA levels. Furthermore, we discovered that METTL3/14-MNK2 axis was up-regulated notably after acute skeletal muscle injury. Collectively, our studies revealed that the m6A writers METTL3/METTL14 and the m6A reader YTHDF1 orchestrate MNK2 expression posttranscriptionally and thus control ERK signaling, which is required for the maintenance of muscle myogenesis and may contribute to regeneration.

Characterization of Long Non-coding RNAs Modified by m6A RNA Methylation in Skeletal Myogenesis.

Proper development of mammalian skeletal muscle relies on precise gene expression regulation. Our previous studies revealed that muscle development is regulated by both mRNA and long non-coding RNAs (lncRNAs). Accumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in various biological processes, making it essential to profile m6A modification on a transcriptome-wide scale in developing muscle. Patterns of m6A methylation in lncRNAs in developing muscle have not been uncovered. Here, we reveal differentially expressed lncRNAs and report temporal m6A methylation patterns in lncRNAs expressed in mouse myoblasts and myotubes by RNA-seq and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs exhibit temporal differential expression, and m6A-lncRNAs harbor the consensus m6A motif "DRACH" along lncRNA transcripts. Interestingly, we found that m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs. Overexpression or knockdown of m6A methyltransferase METTL3 alters the expression levels of these lncRNAs. Furthermore, we highlight that the function of m6A genic lncRNAs might correlate to their nearby mRNAs. Our work reveals a fundamental expression reference of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in developing muscle.

mascRNA and its parent lncRNA MALAT1 promote proliferation and metastasis of hepatocellular carcinoma cells by activating ERK/MAPK signaling pathway.

MALAT1-associated small cytoplasmic RNA (mascRNA) is a cytoplasmic tRNA-like small RNA derived from nucleus-located long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). While MALAT1 was extensively studied and was found to function in multiple cellular processes, including tumorigenesis and tumor progression, the role of mascRNA was largely unknown. Here we show that mascRNA is upregulated in multiple cancer cell lines and hepatocellular carcinoma (HCC) clinical samples. Using HCC cells as model, we found that mascRNA and its parent lncRNA MALAT1 can both promote cell proliferation, migration, and invasion in vitro. Correspondingly, both of them can enhance the tumor growth in mice subcutaneous tumor model and can promote metastasis by tail intravenous injection of HCC cells. Furthermore, we revealed that mascRNA and MALAT1 can both activate ERK/MAPK signaling pathway, which regulates metastasis-related genes and may contribute to the aggressive phenotype of HCC cells. Our results indicate a coordination in function and mechanism of mascRNA and MALAT1 during development and progress of HCC, and provide a paradigm for deciphering tRNA-like structures and their parent transcripts in mammalian cells.

RNA sequencing: new technologies and applications in cancer research.

Over the past few decades, RNA sequencing has significantly progressed, becoming a paramount approach for transcriptome profiling. The revolution from bulk RNA sequencing to single-molecular, single-cell and spatial transcriptome approaches has enabled increasingly accurate, individual cell resolution incorporated with spatial information. Cancer, a major malignant and heterogeneous lethal disease, remains an enormous challenge in medical research and clinical treatment. As a vital tool, RNA sequencing has been utilized in many aspects of cancer research and therapy, including biomarker discovery and characterization of cancer heterogeneity and evolution, drug resistance, cancer immune microenvironment and immunotherapy, cancer neoantigens and so on. In this review, the latest studies on RNA sequencing technology and their applications in cancer are summarized, and future challenges and opportunities for RNA sequencing technology in cancer applications are discussed.

SnoRNAs from the filamentous fungus Neurospora crassa: structural, functional and evolutionary insights.

BACKGROUND: SnoRNAs represent an excellent model for studying the structural and functional evolution of small non-coding RNAs involved in the post-transcriptional modification machinery for rRNAs and snRNAs in eukaryotic cells. Identification of snoRNAs from Neurospora crassa, an important model organism playing key roles in the development of modern genetics, biochemistry and molecular biology will provide insights into the evolution of snoRNA genes in the fungus kingdom. RESULTS: Fifty five box C/D snoRNAs were identified and predicted to guide 71 2'-O-methylated sites including four sites on snRNAs and three sites on tRNAs. Additionally, twenty box H/ACA snoRNAs, which potentially guide 17 pseudouridylations on rRNAs, were also identified. Although not exhaustive, the study provides the first comprehensive list of two major families of snoRNAs from the filamentous fungus N. crassa. The independently transcribed strategy dominates in the expression of box H/ACA snoRNA genes, whereas most of the box C/D snoRNA genes are intron-encoded. This shows that different genomic organizations and expression modes have been adopted by the two major classes of snoRNA genes in N. crassa . Remarkably, five gene clusters represent an outstanding organization of box C/D snoRNA genes, which are well conserved among yeasts and multicellular fungi, implying their functional importance for the fungus cells. Interestingly, alternative splicing events were found in the expression of two polycistronic snoRNA gene hosts that resemble the UHG-like genes in mammals. Phylogenetic analysis further revealed that the extensive separation and recombination of two functional elements of snoRNA genes has occurred during fungus evolution. CONCLUSION: This is the first genome-wide analysis of the filamentous fungus N. crassa snoRNAs that aids in understanding the differences between unicellular fungi and multicellular fungi. As compared with two yeasts, a more complex pattern of methylation guided by box C/D snoRNAs in multicellular fungus than in unicellular yeasts was revealed, indicating the high diversity of post-transcriptional modification guided by snoRNAs in the fungus kingdom.

[Clinical significance of serum levels of endostatin in patients with clear cell renal cell carcinoma].

OBJECTIVE: To assess serum levels of endogenous endostatin in patients with clear cell renal cell carcinoma (CCRCC) and to determine the relationship of these levels to tumor stage, grade. METHODS: From March 2004 to October 2008, preoperative serum were obtained from 138 consecutive patients with CCRCC (73 patients in T1, 39 patients in T2, 20 patients in T3, and 6 patients in T4) and 40 healthy controls. Serum levels of endostatin were measured by sandwich-ELISA. Associations between circulating endostatin levels and clinicopathologic factors and clinical outcome were determined. RESULTS: Endostatin levels did not differ significantly between the patients with CCRCC (93.1 microg/L) and healthy controls (78.9 microg/L, P > 0.05). Serum levels of endostatin were significantly higher in the T2-4 CCRCC patients (107.2 microg/L) than those of the T1 patients (80.4 microg/L, P < 0.01). No significant difference was found in the endostatin levels among the T2-4 patients, or between healthy controls and the T1 patients. The serum endostatin concentration was significantly higher in the metastasis group (118.4 microg/L) than in the no metastasis group (89.5 microg/L, P < 0.05), but there was no significant difference between patients with distant metastasis group (122.0 microg/L) and lymph nodes metastasis (110.0 microg/L, P > 0.05). Patients with G3-4 tumors had significantly higher endostatin levels (111.8 microg/L) than those of patients with G1 (80.4 microg/L) and G2 tumors (86.2 microg/L, P < 0.01), but endostatin levels did not differ significantly between the two groups (P > 0.05). CONCLUSION: Preoperative serum levels of endostatin elevated in patients with CCRCC and associated with higher stage and grade.

Identification of novel chicken microRNAs and analysis of their genomic organization.

MicroRNAs (miRNAs) represent a family of small noncoding RNAs with important regulatory roles in diverse biological processes ranging from cell differentiation to organism development. In chickens, the full set of miRNAs and the expression patterns of miRNAs during development are still poorly understood when compared to the other vertebrates. In this study, we identified 29 novel miRNAs and 140 potential miRNA loci in the chicken genome by combining the experimental and computational analyses. Detailed expression patterns of 49 miRNAs were first characterized by Northern blotting and indicated the cooperativity of the miRNA expression with their function in embryogenesis and organogenesis. Twenty-seven miRNA clusters were systematically evaluated in the chicken genome and diverse expression patterns for closely linked miRNAs were observed. Our results significantly expand the set of known miRNAs in the chicken and provide the basis for understanding the structural and functional evolution of miRNA genes in vertebrates.