Increased miR-203-3p and reduced miR-21-5p synergistically inhibit proliferation, migration, and invasion in esophageal cancer cells.

Dysregulation of miR-203-3p and miR-21-5p has been identified in esophageal cancer (EC). The restoration of miR-203-3p and reduction of miR-21-5p were able to cause tumor suppression. Here, co-transfection of miR-203-3p mimics and miR-21-5p inhibitors led to an extraordinary increased expression of miR-203-3p and synergistically inhibited proliferation, migration, and invasion in EC cells. Moreover, we found that Ran GTPase (Ran) was dramatically inhibited in EC cells treated with the co-transfection of miR-203-3p mimics and miR-21-5p inhibitors. Finally, in-vivo studies showed that overexpression of miR-203-3p, combined with the suppression of miR-21-5p, significantly co-inhibited growth of tumors. The obtained data suggested that the development of miRNA-based combination therapeutics represents a promising cancer treatment strategy.

Knockdown of Ran GTPase expression inhibits the proliferation and migration of breast cancer cells.

Breast cancer is the second leading cause of cancer­associated mortality in women worldwide. Strong evidence has suggested that Ran, which is a small GTP binding protein involved in the transport of RNA and protein across the nucleus, may be a key cellular protein involved in the metastatic progression of cancer. The present study investigated Ran gene expression in breast cancer tissue samples obtained from 140 patients who had undergone surgical resection for breast cancer. Western blot analysis of Ran in breast cancer tissues and paired adjacent normal tissues showed that expression of Ran was significantly increased in breast cancer tissues. Immunohistochemistry analyses conducted on formalin­fixed paraffin­embedded breast cancer tissue sections revealed that Ran expression was associated with tumor histological grade, nerve invasion and metastasis, vascular metastasis and Ki­67 expression (a marker of cell proliferation). Kaplan­Meier survival analysis showed that increased Ran expression in patients with breast cancer was positively associated with a poor survival prognosis. Furthermore, in vitro experiments demonstrated that highly migratory MDA­MB­231 cancer cells treated with Ran­si­RNA (si­Ran), which knocked down expression of Ran, exhibited decreased motility in trans­well migration and wound healing assays. Cell cycle analysis of Ran knocked down MDA­MB­231 cells implicated Ran in cell cycle arrest and the inhibition of proliferation. Furthermore, a starvation and re­feeding (CCK­8) assay was performed, which indicated that Ran regulated breast cancer cell proliferation. Taken together, the results provide strong in vitro evidence of the involvement of Ran in the progression of breast cancer and suggest that it could have high potential as a therapeutic target and/or marker of disease.

RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2.

Several microsatellite-expansion diseases are characterized by the accumulation of RNA foci and RAN proteins, raising the possibility of a mechanistic connection. We explored this question using myotonic dystrophy type 2, a multisystemic disease thought to be primarily caused by RNA gain-of-function effects. We demonstrate that the DM2 CCTG⋅CAGG expansion expresses sense and antisense tetrapeptide poly-(LPAC) and poly-(QAGR) RAN proteins, respectively. In DM2 autopsy brains, LPAC is found in neurons, astrocytes, and glia in gray matter, and antisense QAGR proteins accumulate within white matter. LPAC and QAGR proteins are toxic to cells independent of RNA gain of function. RNA foci and nuclear sequestration of CCUG transcripts by MBNL1 is inversely correlated with LPAC expression. These data suggest a model that involves nuclear retention of expansion RNAs by RNA-binding proteins (RBPs) and an acute phase in which expansion RNAs exceed RBP sequestration capacity, are exported to the cytoplasm, and undergo RAN translation. VIDEO ABSTRACT.

RanBP1 governs spindle assembly by defining mitotic Ran-GTP production.

Accurate control of the Ras-related nuclear protein (Ran) GTPase cycle depends on the regulated activity of regulator of chromosome condensation 1 (RCC1), Ran's nucleotide exchange factor. RanBP1 has been characterized as a coactivator of the Ran GTPase-activating protein RanGAP1. RanBP1 can also form a stable complex with Ran and RCC1, although the dynamics and function of this complex remain poorly understood. Here, we show that formation of the heterotrimeric RCC1/Ran/RanBP1 complex in M phase Xenopus egg extracts controls both RCC1's enzymatic activity and partitioning between the chromatin-bound and soluble pools of RCC1. This mechanism is critical for spatial control of Ran-guanosine triphosphate (GTP) gradients that guide mitotic spindle assembly. Moreover, phosphorylation of RanBP1 drives changes in the dynamics of chromatin-bound RCC1 pools at the metaphase-anaphase transition. Our findings reveal an important mitotic role for RanBP1, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring accurate execution of Ran-dependent mitotic events.

Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins.

Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3.

Ran GTPase induces EMT and enhances invasion in non-small cell lung cancer cells through activation of PI3K-AKT pathway.

Ras-related nuclear protein (Ran) GTPase is upregulated in non-small cell lung cancer (NSCLC) cells and is required for NSCLC cell survival. However, the effect of Ran on NSCLC cell invasion and epithelial to mesenchymal transition (EMT) remains unclear. This study found that Ran expression was much higher in highly invasive NSCLC cells than in lowly invasive NSCLC cells. Ectopic expression of Ran enhanced invasion and induced EMT in NSCLC cells. Inhibition of the PI3K-AKT pathway by LY294002, but not the MEK-ERK pathway by PD98509, reversed the above effects in these cells induced by Ran overexpression. In conclusion, our findings demonstrate that Ran induces EMT and enhances invasion in NSCLC cells through the activation of PI3K-AKT signaling. Thus, Ran may be a potential target for NSCLC therapeutic intervention.

High Ran level is correlated with poor prognosis in patients with colorectal cancer.

BACKGROUND: The Ras-like nuclear protein (Ran) is involved in the regulation of nuclear transport, microtubule nucleation and dynamics, and spindle assembly. Its fundamental function is nucleocytoplasmic transport of RNA and proteins. The expression and potential role of Ran in colorectal cancer (CRC) remain unclear. The aim of this study was to investigate the relationship between Ran expression and CRC characteristics. The potential role of Ran as a prognostic indicator was also evaluated. METHODS: We used immunohistochemistry and western blotting to detect Ran expression in 287 CRC tissues. The relationships between Ran expression and clinicopathological characteristics and overall survival rate were statistically analyzed. RESULTS: CRC tissues had significantly higher Ran expression than normal colorectal epithelial cells. Ran was positively correlated with depth of invasion, lymph node metastases, distant metastases, tumor differentiation, and tumor-node-metastasis stage. However, no correlation was found between Ran expression and patient age or sex. The overall survival rate was consistently and significantly lower in patients with Ran-positive tumors than in those with Ran-negative tumors. CONCLUSION: Our findings emphasize the important role of Ran in differentiation, disease stage, and metastasis in human CRC. Ran may play an important role in the development of CRC and may serve as a novel prognostic indicator of CRC.

Tumor cell dependence on Ran-GTP-directed mitosis.

Deregulated cell division is a hallmark of cancer, but whether tumor cells become dependent on specific mitotic mechanisms is not known. Here, we show that the small GTPase Ran, a regulator of mitotic spindle formation, is differentially overexpressed in human cancer as compared with normal tissues, in vivo. Acute silencing of Ran in various tumor cell types causes aberrant mitotic spindle formation, mitochondrial dysfunction, and apoptosis. This pathway does not require p53, Bax, or Smac, but is controlled by survivin as a novel Ran target in cancer. Conversely, loss of Ran in normal cells is well tolerated and does not result in mitotic defects or loss of cell viability. Therefore, tumor cells can become dependent on Ran signaling for cell division, and targeting this pathway may provide a novel and selective anticancer strategy.

Phosphorylation of RCC1 in mitosis is essential for producing a high RanGTP concentration on chromosomes and for spindle assembly in mammalian cells.

Spindle assembly is subject to the regulatory controls of both the cell-cycle machinery and the Ran-signaling pathway. An important question is how the two regulatory pathways communicate with each other to achieve coordinated regulation in mitosis. We show here that Cdc2 kinase phosphorylates the serines located in or near the nuclear localization signal (NLS) of human RCC1, the nucleotide exchange factor for Ran. This phosphorylation is necessary for RCC1 to generate RanGTP on mitotic chromosomes in mammalian cells, which in turn is required for spindle assembly and chromosome segregation. Moreover, phosphorylation of the NLS of RCC1 is required to prevent the binding of importin alpha and beta to RCC1, thereby allowing RCC1 to couple RanGTP production to chromosome binding. These findings reveal that the cell-cycle machinery directly regulates the Ran-signaling pathway by placing a high RanGTP concentration on the mitotic chromosome in mammalian cells.

Sperm membrane protein (hSMP-1) and RanBPM complex in the microtubule-organizing centre.

hSMP-1 is a human sperm membrane protein expressed during development. It is a testis-specific component produced during male germ cell differentiation. Proteins that interact with hSMP-1 were identified by the application of the yeast two-hybrid system. One of the components, RanBPM, was found to be associated with hSMP-1 under both in vitro and in vivo conditions. In the human testis, RanBPM is produced in spermatogonia and primary spermatocytes, suggesting expression during the early stages of spermatogenesis; whereas in the rat testis, it is located in round and elongated spermatids, similar to hSMP-1, suggesting expression of both components during spermiogenesis. Images obtained by immunofluorescence and confocal scanning microscopy of CHO-K1 cells co-transfected with pEGFP-C1-hSMP-1 and pDsRed1-Nl-RanBPM revealed that RanBPM and hSMP-1 are distributed in discrete loci throughout the cytoplasm. When superimposed, the stained spots appeared as congruent yellow areas, indicative of co-localization and probable complex formation of these two components. This interaction between hSMP-1 and RanBPM may be involved in the process of male germ cell differentiation. In CHO-Kl cells transfected with pEGFP-Cl-hSMP-1, the exogenously expressed hSMP-1 was found to co-localize with alpha-tubulin. Depolymerization of microtubules can be induced in CHO-Kl cells by cold treatment. In cells transfected with the pEGFP-Cl vector, the dispersed tubulins promptly reassembled upon warming. However, in cells transfected with pEGFP-Cl-hSMP-1, reassembly of the dispersed tubulins was blocked even upon rewarming of the cells. These findings suggest that hSMP-1 interacts with tubulins and thereby may modulate microtubule assembly and/or activity.

A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo.

The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1-Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.

Ran GTPase expression during early development of the mouse embryo.

Ran is a small GTP-binding protein involved in several essential roles for cell viability. This observation implies Ran might be ubiquitously expressed during development. However, Ran shows a differentiated expression pattern that is restricted to specific tissues from embryo to adult. At early embryonic stages of mouse development we found persistent Ran expression in proliferating neural tissue, neural crest derived dorsal root ganglions and sensory pits. We also showed an accumulation of Ran transcripts in main embryonic haematopoietic tissues: blood islands first and then hepatic bud. In advanced stages of development Ran is also expressed in other tissues showing a high cell turnover.

Hypothesis: Ran GTPase-based potential therapeutic interventions against lethal microbial infections.

Host innate immune response represents a vital immediate defense against infections by a diverse group of microorganisms that include bacteria, viruses, and fungi. Many types of cell surface receptors in mammalian cells specifically recognize particular groups of microorganisms and transmit response signals to the nuclei via multiple signal transduction pathways. These signaling pathways must merge at some point and are likely to be redundant, as the host innate immune response to many microorganisms is remarkably similar; it is characterized by the production of proinflammatory cytokines such as TNFalpha, IL-1, and IL-6 by the principal cell types--macrophages and dendritic cells. Since these cytokines influence greatly the magnitude of the cascade of inflammatory events, the proportion and the actual amount of each among the cytokine group may be a characteristic of each type of infections. Immune modulation by systematically up-regulate or down-modulate these cytokines would conceivably have major therapeutic potential. We have recently shown that two alleles of Ran cDNAs--RanT/n and RanC/d--may possess these characteristics. Thus the application of Ran to the treatment of septic shock, lethal anthrax shock, or adenovirus-induced toxicities may open up many interesting possibilities in the future.

The involvement of Ran GTPase in lipopolysaccharide endotoxin-induced responses.

By functional cloning, we have established that Ran GTPase is involved in LPS-induced signal transduction. This has been accomplished by several functional comparisons of the two cDNAs, Lps(n)/Ran (or RanT/n) and Lps(d)/Ran (or RanC/d), which were isolated from cDNA libraries of LPS responder and hyporesponder mice, respectively. The letter n refers to the "normal" phenotype and the letter d refers to the "deficient" phenotype. Consistent with our previous results, more animal studies indicated that adenoviral transduction of RanC/d cDNA, but not RanT/n cDNA, into sensitive mice conferred significant resistance against endotoxin challenge. Thus the incorporation of RanC/d cDNA into gene therapy protocols as a therapeutic sequence remains very attractive. At steady state, hematopoietic cells transduced with RanC/d cDNA led to about a 10-fold increase in exogenous Ran protein compared with RanT/n cDNA. Furthermore, our cumulative data suggest that a slight elevation of Ran protein in B cells enhances LPS responsiveness, but the same elevation of Ran in macrophages does not. On the other hand, a high level of overexpression of Ran in both macrophages and B cells down-regulates LPS signal transduction. Thus LPS-induced signal transduction in macrophages and B cells is likely to occur via different signaling pathways.

Identification of a novel putative Ran-binding protein and its close homologue.

In the process of cloning genes at the breakpoint of t(5;14) (q34;q11), a recurring translocation in acute lymphoblastic leukemia, we isolated and characterized a novel gene at 5q34, and a close human homologue (66% amino acid identity) located at 8p11-12. The presence of an importin-beta N-terminal domain at their N-terminus, their size of approximately 110 kD, their nuclear localization and the identity of the homologue to a gene of a recently submitted RanGTP binding protein (RanBP16), suggest that its protein is a novel member of the importin-beta superfamily of nuclear transport receptors, therefore called RanBP17. Northern blot analysis of human tissues revealed a ubiquitous expression pattern of the RanBP16 gene and a very restricted expression pattern of the RanBP17 gene, showing high expression in testis and pancreas. Both genes are evolutionary conserved and show a high (99 and 94%) amino acid conservation with their murine counterparts and a striking similarity (40%) to a protein product of Caenorhabditis elegans (C35A5.8).

Expression analysis of human HL60 cells exposed to 60 Hz square- or sine-wave magnetic fields.

A total of 960 complementary DNA (cDNA) clones from an HL60 cell cDNA library were screened to discover genes that were differentially expressed in HL60 cells exposed to 60 Hz square-wave magnetic fields (MFs) compared to sham-exposed cells. Square-wave fields are rich in odd harmonic frequency content. We used a two-gel cDNA library screening method (BIGEL) to identify treatment-induced alterations in gene expression. Four cDNA clones were tentatively identified as differentially expressed after exposure to square-wave MFs at 2 mT for 24 h. BIGEL-identified genes (GenBank accession number) corresponding to these clones were: TI227H (D50525), EST Homo sapiens partial cDNA (Z17814), human ribosomal protein S13 (L01124), and AICAR transformylase mRNAs (D82348). The differences in mRNA levels were not confirmed in test compared to experimental cells by Northern analysis. In other experiments, we used concurrent exposure to 60 Hz sine- or square-wave MFs (0 or 2 mT, duration of 3 or 24 h, no postexposure delay). In addition to the four BIGEL genes, we also investigated MYC, HSP70, RAN and SOD1. In the case of MYC and HSP70, square-wave MFs appeared to exhibit more marked alterations when compared to sinusoidal waveforms, but the overall results indicated no effect of possible differential magnetic-field-induced expression of all eight genes. In contrast, alterations of mRNA levels were observed for seven genes after exposure to X irradiation, hyperthermia and TPA. These results are contrary to previously proposed similarities between the action of these agents and MF effects on gene transcription.

Elevated levels of RanBP7 mRNA in colorectal carcinoma are associated with increased proliferation and are similar to the transcription pattern of the proto-oncogene c-myc.

We have used suppression subtractive hybridisation, "in silico" cloning and reverse Northern dot blot analysis to identify significant up-regulation of RanBP7 transcription in a human colorectal carcinoma. Quantitative RT-PCR analyses using the Taqman system demonstrated that RanBP7 mRNA levels were elevated in 47/75 colorectal tumours. There was no significant difference in 17 matched normal and tumour pairs and reduced levels in 11. Since RanBP7 specifies a key member of nuclear transport receptors responsible for the nuclear import of histone H1 and ribosomal proteins, we investigated whether this up-regulation might be proliferation-associated. RanBP7 mRNA copy numbers were significantly correlated with those of proliferating cell nuclear antigen in both normal and cancer tissue. Interestingly, the transcription pattern of the proto-oncogene c-myc showed a similar correlation with PCNA mRNA. Our results highlight the need for the careful interpretation of quantitative data that compare mRNA levels in normal and cancer tissue.

Crystallization and preliminary X-ray diffraction analysis of the Saccharomyces cerevisiae ran-binding protein Mog1p.

Mog1p binds the Ras-family GTPase Ran/Gsp1p, which has a central role in nucleocytoplasmic transport and cell-cycle progression. Overexpression of MOG1 is able to suppress temperature-sensitive gsp1 mutants in yeast; Deltamog1 null mutants display temperature-sensitive defects in nuclear trafficking. Orthorhombic crystals of bacterially expressed Mog1p that diffract to beyond 2 A resolution using synchrotron radiation have been obtained. The crystals have P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 39.67, b = 51.96, c = 112.23 A, a Matthews coefficient of 2.44 A(3) Da(-1), an estimated solvent content of 49.5% and one chain in the asymmetric unit.