MicroRNA-210 regulates cancer cell proliferation through targeting fibroblast growth factor receptor-like 1 (FGFRL1)

The importance of microRNAs (miRNAs) in human malignancies has been well recognized. Here, we report that the expression of microRNA-210 (miR-210) is down-regulated in human esophageal squamous cell carcinoma and derived cell lines. Marked decreases in the level of miR-210 were observed especially in poorly differentiated carcinomas. We found that miR-210 inhibits cancer cell survival and proliferation by inducing cell death and cell cycle arrest in G(1)/G(0) and G(2)/M. Finally, we identified fibroblast growth factor receptor-like 1 (FGFRL1) as a target of miR-210 in esophageal squamous cell carcinoma and demonstrated that FGFRL1 accelerates cancer cell proliferation by preventing cell cycle arrest in G(1)/G(0). Taken together, our findings show an important role for miR-210 as a tumor-suppressive microRNA with effects on cancer cell proliferation.     

Elevation of highly up-regulated in liver cancer (HULC) by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18

Long noncoding RNAs (lncRNAs) play crucial roles in human cancers. It has been reported that lncRNA highly up-regulated in liver cancer (HULC) is dramatically up-regulated in hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) contributes importantly to the development of HCC. However, the function of HULC in HCC mediated by HBx remains unclear. Here, we report that HULC is involved in HBx-mediated hepatocarcinogenesis. We found that the expression levels of HULC were positively correlated with those of HBx in clinical HCC tissues. Moreover, we revealed that HBx up-regulated HULC in human immortalized normal liver L-O2 cells and hepatoma HepG2 cells. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay showed that HBx activated the HULC promoter via cAMP-responsive element-binding protein. We further demonstrated that HULC promoted cell proliferation by methyl thiazolyl tetrazolium, 5-ethynyl-2'-deoxyuridine, colony formation assay, and tumorigenicity assay. Next, we hypothesized that HULC might function through regulating a tumor suppressor gene p18 located near HULC in the same chromosome. We found that the mRNA levels of p18 were inversely correlated with those of HULC in the above clinical HCC specimens. Then, we validated that HULC down-regulated p18, which was involved in the HULC-enhanced cell proliferation in vitro and in vivo. Furthermore, we observed that knockdown of HULC could abolish the HBx-enhanced cell proliferation through up-regulating p18. Thus, we conclude that the up-regulated HULC by HBx promotes proliferation of hepatoma cells through suppressing p18. This finding provides new insight into the roles of lncRNAs in HBx-related hepatocarcinogenesis.     

食管鳞状细胞癌中血管生成拟态及MMP-1蛋白的表达

目的 研究人食管鳞状细胞癌(human esophageal squamous cell carcinoma,ESCC)中是否存在血管生成拟态(vasculogenic mimicry,VM)及其与(Matrix metalloproteinase-1,MMP-1)表达的关系,以及MMP-1过表达的临床意义.方法收集118例食管鳞状细胞癌的标本,每例均有完整的临床资料,利用CD31(Platelet endothelial cell adhesion molecule,PECAM-1)和PAS套染观察是否存在VM,对VM组和对照组进行MMP-1染色,分析VM与MMP-1表达的关系及MMP-1表达与临床病理学参数的关系.结果 118例食管鳞状细胞癌组织中有34例(28.81%)存在VM,有VM生成组的MMP-1过表达比例显著高于无VM组,两组之间的差异有统计学意义(P<0.05).有淋巴结转移组的MMP-1过表达的比例显著高于无淋巴结转移组(P<0.05);浸润至深肌层及外膜层的MMP-1过表达的比例显著高于浸润之粘膜层和浅肌层的(P<0.05);临床分期Ⅲ-Ⅳ期的MMP-1蛋白的过表达显著高于临床分期Ⅰ-Ⅱ期的(P<0.05).结论 食管鳞状细胞癌中存在VM,MMP-1的过表达可能促进VM形成;VM的存在和MMP-1过表达共同促进淋巴结转移和肿瘤浸润.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs

Cancer cells secreting excess latent TGF-β are often resistant to TGF-β induced growth inhibition. We observed that RNAi against TGF-β1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-β signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression. The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively. Our data suggest a novel mechanism in which latent TGF-β1, thrombospondin 1, and furin form a microRNA-mediated regulatory feedback loop. For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.     

Involvement of polo-like kinase 1 (Plk1) in mitotic arrest by inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase-ribosomal S6 kinase 1 (MEK-ERK-RSK1) cascade

Cell division is controlled through cooperation of different kinases. Of these, polo-like kinase 1 (Plk1) and p90 ribosomal S6 kinase 1 (RSK1) play key roles. Plk1 acts as a G(2)/M trigger, and RSK1 promotes G(1) progression. Although previous reports show that Plk1 is suppressed by RSK1 during meiosis in Xenopus oocytes, it is still not clear whether this is the case during mitosis or whether Plk1 counteracts the effects of RSK1. Few animal models are available for the study of controlled and transient cell cycle arrest. Here we show that encysted embryos (cysts) of the primitive crustacean Artemia are ideal for such research because they undergo complete cell cycle arrest when they enter diapause (a state of obligate dormancy). We found that Plk1 suppressed the activity of RSK1 during embryonic mitosis and that Plk1 was inhibited during embryonic diapause and mitotic arrest. In addition, studies on HeLa cells using Plk1 siRNA interference and overexpression showed that phosphorylation of RSK1 increased upon interference and decreased after overexpression, suggesting that Plk1 inhibits RSK1. Taken together, these findings provide insights into the regulation of Plk1 during cell division and Artemia diapause cyst formation and the correlation between the activity of Plk1 and RSK1.     

PinX1, a Telomere Repeat-binding Factor 1 (TRF1)-interacting Protein,Maintains Telomere Integrity by Modulating TRF1 Homeostasis,the Process in Which Human Telomerase Reverse Transcriptase (hTERT) Plays Dual Roles

TRF1, a telomere-binding protein, is important for telomere protection and homeostasis. PinX1 interacts with TRF1, but the physiological consequences of their interaction in telomere protection are not yet understood. Here we investigated PinX1 function on TRF1 stability in HeLa cells. PinX1 overexpression stabilized TRF1, but PinX1 depletion by siRNA led to TRF1 degradation, TRF1 ubiquitination, and less TRF1 telomere association. The depletion also induced DNA damage responses at telomeres and chromosome instability. These telomere dysfunctional phenotypes were in fact due to TRF1 deficiency. We also report that hTERT, a catalytic component of telomerase, plays dual roles in the TRF1 steady state pathway. PinX1-mediated TRF1 stability was not observed in hTERT-negative immortal cells, but was pronounced when hTERT was ectopically expressed in the cells, suggesting that hTERT may be needed in the PinX1-mediated TRF1 stability pathway. Interestingly, the knockdown of both PinX1 and hTERT in HeLa cells stabilized TRF1, suppressed DNA damage response activation, and restored chromosome stability. In summary, our findings suggested that PinX1 may maintain telomere integrity by regulating TRF1 stability and that hTERT may act as both a positive and a negative regulator of TRF1 homeostasis in a PinX1-dependent manner.     

Genome-wide Approaches Reveal Functional Vascular Endothelial Growth Factor (VEGF)-inducible Nuclear Factor of Activated T Cells (NFAT) c1 Binding to Angiogenesis-related Genes in the Endothelium

VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.     

Disruption of the ATP-binding cassette B7 (ABTM-1/ABCB7) induces oxidative stress and premature cell death in Caenorhabditis elegans

X-linked sideroblastic anemia with ataxia (XLSA/A) is a rare inherited disorder characterized by mild anemia and ataxia. XLSA/A is caused by mutations in the ABCB7 gene, which encodes a member of the ATP-binding cassette transporter family. Studies in yeast, mammalian cells, and mice have shown that ABCB7 functions in the transport of iron-sulfur (Fe-S) clusters into the cytoplasm. To further investigate the mechanism of this disease, we have identified and characterized the Caenorhabditis elegans homologue of the ABCB7 gene, abtm-1. We have studied the function of abtm-1 using mutants and RNAi. abtm-1-depleted animals produce arrested embryos that have morphogenetic defects and unusual premature, putative apoptotic events. abtm-1(RNAi) animals also show accumulation of ferric iron and increased oxidative stress. Despite the increased level of oxidative stress in abtm-1(RNAi) animals, they have an increased life span. We observed accumulation of DAF-16/FOXO in the nuclei of affected animals and elevation of the expression of SOD-3, a well established target of DAF-16, which may explain the increased life span extension of these animals. abtm-1 is strongly expressed in tissues with a high energy demand, and abtm-1(RNAi) animals have phenotypes that reflect the need for abtm-1 in these tissues. Finally, we show that reducing the function of other genes involved in Fe-S cluster production produces similar phenotypic consequences to abtm-1 loss of function. Therefore, ablation of abtm-1 in C. elegans provides a model in which to investigate the mechanism underlying XLSA/A.