Myc target gene, long intergenic noncoding RNA, Linc00176 in hepatocellular carcinoma regulates cell cycle and cell survival by titrating tumor suppressor microRNAs.

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities for medical intervention. Recent evidence has accumulated that long noncoding RNAs (lncRNAs) are important regulators of disease processes including cancer. Chromatin remodeling in cancer cells may result in an unusual expression of lncRNAs and indeed it has been shown that more than 7000 unannotated lncRNAs are expressed in HCCs. We identified a novel long intergenic noncoding RNA, Linc00176, that plays a role in proliferation and survival of HCC. Linc00176 regulates expression of more than 200 genes by the sponge function for tumor suppressor miRNAs, miR-9 and miR-185. Linc00176 is expressed at a high level only in HCC, and is activated by Myc, Max and AP-4 transcription regulators. Myc also upregulates miR-9 and miR-185. In Linc00176-depleted HCC, these miRNAs were released from Linc00176 and downregulated their target mRNAs. Thus, depletion of Linc00176 disrupted the cell cycle and induced necroptosis in HCC via released tumor suppressor miRNAs. These data indicate that atypically expressed lncRNAs may be useful targets for cancer therapy.

Correction to: mouse mammary tumour virus (MMTV) and human breast cancer with neuroendocrine differentiation.

[This corrects the article DOI: 10.1186/s13027-017-0135-8.].

Depletion of three combined THOC5 mRNA export protein target genes synergistically induces human hepatocellular carcinoma cell death.

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy.

Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation.

Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

A pathway from leukemogenic oncogenes and stem cell chemokines to RNA processing via THOC5.

THOC5 is a member of the THO complex that is involved in processing and transport of mRNA. We have shown previously that hematopoietic stem cells have an absolute requirement for THOC5 for survival and that THOC5 is phosphorylated on tyrosine 225 as a consequence of leukemogenic protein tyrosine kinase (PTK) action. We have investigated pathways for THOC5 phosphorylation to develop an understanding of THO complex modulation by tyrosine kinase (TK) oncogenes in leukemias. We demonstrate that THOC5 phosphorylation is mediated by Src PTK and CD45 protein tyrosine phosphatase action and that this event is sensitive to oxidative status. We show that THOC5 phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia (CML) and that this phosphorylation is sensitive to the frontline drugs used in CML treatment. Further we show that THOC5 Y225 phosphorylation governs mRNA binding. In addition, CXCL12 is shown to induce THOC5 Y225 phosphorylation, and site-directed mutagenesis demonstrates that this modulates motile response. In conclusion, we delineate a signaling pathway stimulated by leukemogenic PTKs, chemokines and oxidative stress that can affect THO complex mediation of gene expression describing mechanisms for post-transcriptional regulation of protein levels.

Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer.

BACKGROUND: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. AIM: To investigate the possibility of an association between breast cancer and MMTV-like viruses. METHODS: Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV-associated mammary tumours from C3H experimental mice. The presence of MMTV-like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. RESULTS: MMTV-like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV-associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV-like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. CONCLUSION: Some human breast cancer specimens, in which MMTV-like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV-associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an association between the presence of MMTV-like env DNA sequences and some human breast cancers.

Hormone replacement therapy use dramatically increases breast oestrogen receptor expression in obese postmenopausal women.

BACKGROUND: It is known that use of hormone replacement therapy (HRT) by postmenopausal women increases the risk of breast cancer. METHOD: In this study, oestrogen receptor (ER)-alpha expression is examined using standard immunoperoxidase technique. RESULTS: Normal breast samples of 11 Australian postmenopausal women have been included in the ER-alpha study; the result showed a strong correlation (r(2) = 0.80) between ER-alpha expression in normal breast epithelial cells and body mass index (BMI) in normal women who currently use HRT. CONCLUSION: This finding confirms that the possibility of increased risk of breast cancer associated with increased ER-alpha expression in normal breast epithelial cells, in turn associated with high BMI and the use of HRT.

Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase.

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.

Striatal glutamate antagonism induces contralateral neglect.

To assess the role of striatal glutamatergic synapses in mediating sensorimotor orientation behavior, glutamate receptor antagonists were infused into the left striatum of awake rats and behavioral orientation to contralateral and ipsilateral stimuli were quantified. The AMPA-kainate antagonist, DNQX, and the NMDA antagonist, CPP, both induced a large asymmetry in responding, such that the rats oriented much less to stimuli presented contralateral to the antagonist infusions. Furthermore, intrastriatal glutamate antagonist infusions increased the occurrence of incorrect responses, or turning away from a contralaterally-presented stimulus. In a separate experiment, intrastriatal DNQX was shown to block kainic acid (KA)-induced Fos expression in the striatum, but not in adjacent cerebral cortex, suggesting that the diffusion of this drug is restricted to the striatum.

Alzheimer disease: improved visual interpretation of PET images by using three-dimensional stereotaxic surface projections.

PURPOSE: To compare the diagnostic usefulness of three-dimensional (3D) stereotaxic surface projection (SSP) with that of standard transaxial display in brain positron emission tomography (PET) in Alzheimer disease (AD). MATERIALS AND METHODS: Standard transaxial section display and 3D-SSP PET image sets obtained after administration of 2-deoxy-2-[fluorine-18]fluoro-D-glucose in 39 patients with probable AD (aged 53-82 years; 15 men, 24 women) and 40 subjects without AD (aged 21-78 years; 14 men, 26 women) were randomly interpreted. Receiver operating characteristic (ROC) analysis was performed. RESULTS: Diagnostic performance was superior with 3D SSP (Az[section]=0.94,Az[3D SSP]=0.99[Az=area under the ROC curve];P=.043). With 3D SSP, diagnosis of AD was equally good in beginners and experts. The sensitivity and specificity in questionable or mild dementia were 94% and 99% with 3D SSP and 79% and 88% with standard transaxial display. CONCLUSION: Accuracy of detecting AD was improved in PET with 3D SSP.

Serum nitrate levels in ulcerative colitis and Crohn's disease.

BACKGROUND: Nitric oxide is an important mediator in inflammatory and autoimmune-mediated tissue destruction and may be of pathophysiologic importance in inflammatory bowel disease. We studied whether serum levels of nitrate, the stable end-product of nitric oxide, are increased in active Crohn's disease or ulcerative colitis, in comparison with quiescent disease and healthy controls. The setting was the gastroenterology unit of the Free University Hospital, Amsterdam. METHODS: In 146 patients--75 with ulcerative colitis and 71 with Crohn's disease--and 33 controls serum nitrate was measured by the Griess reaction after enzymatic conversion of nitrate to nitrite with nitrate reductase. RESULTS: Median serum nitrate concentrations did not differ statistically significantly between ulcerative colitis (median, 34.2 mumol/l; range, 15.6-229.4 mumol/l), Crohn's disease (median 32.3 mumol; range 13.2-143.2 mumol/l), and healthy controls (median, 28.7 mumol/l; range, 13.0-108.4 mumol/l). However, when active ulcerative colitis patients (median, 44 mumol/l; range, 29.1-229.4 mumol/l were compared with inactive ulcerative colitis patients (median, 31.2 mumol/l; range, 15.6-59.7 mumol/l), a significant difference in nitrate concentration was found (p < 0.0001). A significant positive correlation was found between serum nitrate levels in ulcerative colitis and erythrocyte sedimentation rate (ESR) (r = 0.30, p - 0.01), leucocyte count (r = 0.27, p = 0.02), and thrombocyte count (r = 0.24, p = 0.04). Comparing active Crohn's disease patients (median, 37.5 mumol/l; range, 13.2-143.2 mumol/l) with inactive Crohn's disease patients (median, 31.3 mumol/l; range, 14.5-92.3 mumol/l) also showed a significant difference in serum nitrate concentration (p < 0.009). Serum nitrate levels correlated with the ESR (r = 0.26, p = 0.028) and serum albumin (r = 0.38, p = 0.004) as well. CONCLUSION: Nitric oxide production is increased in both active ulcerative colitis and Crohn's disease and may be implicated in the pathogenesis of inflammatory bowel disease.

Does additional treatment with fish oil mitigate the side effects of recombinant human erythropoietin in dialysis patients?

Since fish oil has been reported to reduce platelet aggregability, to reduce blood viscosity by increasing red blood cell deformability and to lower blood pressure, we studied the effect of dietary supplementation with fish oil on the occurrence of adverse effects in patients receiving recombinant human erythropoietin (rHuEPO). In a prospective, randomized, double blind cross-over design we studied the effect of daily ingestion of 3 g fish oil versus 3 g corn oil (placebo) for 5 months, with a wash-out period of 3 months in between. Thirty-two dialysis patients newly treated with rHuEPO participated. rHuEP0 was given using a low and slow dose regimen (25 U/kg twice weekly s.c.). Target Hct was 35%. Blood pressure, red blood cell deformability, plasma viscosity, fatty acid composition of plasma phospholipids, and fibrinogen levels were measured at 0, 5, 8 and 13 months. In both groups a stable target Hct (35%) was reached within 3 months. Blood pressure was not significantly different between the groups at any time point. In 4 patients (2 on fish oil and 2 on placebo) antihypertensives had to be increased to regulate blood pressure adequately, whereas shunt occlusion occurred in one patient on placebo. Despite a significant increase in the omega-3 fatty acid content of plasma phospholipids during ingestion of fish oil, no significant changes in red blood cell deformability were observed. Since hypertension and shunt occlusion occurred at rates comparable to those reported in the literature, long-term ingestion of fish oil does not appear to mitigate the side effects of low and slow dose rHuEPO.

Diagnosing intracranial vasculitis: the roles of MR and angiography.

PURPOSE: To describe our experience with MR and angiography in diagnosing intracranial vasculitis and to test the hypothesis that MR can be used to screen for patients unlikely to have vasculitis. METHODS: Ninety-two patients who had angiography with "exclude vasculitis" as the indication or who had angiography and a clinical diagnosis of vasculitis were identified. Angiograms of all 92 patients and the MRs of the 70 patients who had both studies were reviewed. RESULTS: Eleven patients had intracranial vasculitis. Angiography showed characteristic changes in 8. MR, performed in 9 of 11 vasculitis cases, was significantly abnormal in all 9. Among 70 patients who had both studies, 19 had MR that was completely normal or showed only incidental findings. None of these 19 was diagnosed with vasculitis. The diagnostic yield of angiography performed to exclude vasculitis was only 6%. CONCLUSIONS: Evaluation for intracranial vasculitis should include MR. A negative MR excludes intracranial vasculitis more definitively than does a negative angiogram and makes the likelihood of finding vasculitis with angiography negligible.

Acute renal failure in patients with severe civilian trauma.

We reviewed our experience with patients suffering from civilian trauma to identify risk factors for the development of acute renal failure (ARF) and ARF outcome. Of the 437 patients consecutively admitted to a surgical intensive care unit (SICU), 206 had a SICU stay of at least 1 day and ARF developed in 30 of these patients. All ARF patients had additional organ system failure (OSF). Pre-existing chronic disease (including chronic renal failure), malnutrition, injury severity score (ISS), number of organs injured, sepsis, and all OSFs before the onset of ARF were factors predisposing to ARF. Mortality was 40%. Chronic disease, malnutrition, ISS, failure of cardiovascular, pulmonary, hepatic, and neurological systems (either before and after ARF) were significantly associated with mortality. When OSFs were considered in their temporal relationships to ARF, only cardiovascular and pulmonary failure before, and gastrointestinal failure after, the onset of ARF were related to mortality. An increasing number of OSFs increased mortality, both before and after the development of ARF. However, the number of OSFs before was significantly greater than after ARF. Sepsis was not associated with increased mortality. Thus, the outcome of ARF patients with critical trauma seems to be dependent on factors predisposing to ARF. Our results suggest that more attention must be paid to prevention of these precipitating conditions.

Factors related to multiple organ system failure and mortality in a surgical intensive care unit.

We retrospectively studied the relative contribution of factors related to the extent of multiple organ system failure (MOSF) and mortality, using multivariate methods to account for the interactions between studied factors, in 538 consecutive patients admitted to a surgical intensive care unit during a 1-year period. MOSF (MOSF score > or = 5) occurred in 88 (16%) of patients. Multiple linear regression selected advancing age, malnutrition, APACHE II score, shock and coma on admission, number of blood transfusions, use of H2 receptor antagonists or antacids, bacteraemia and intra-abdominal infection as independent factors related to the MOSF score. MOSF mortality was 52% and was a major cause of death in critically ill surgical patients. Multiple logistic regression selected advancing age, malnutrition, bacteraemia, APACHE II and MOSF score as major predictors of mortality. Advancing age, malnutrition, shock and coma on admission, transfusion requirement and use of H2 receptor antagonists or antacids may impair host defences of the gastrointestinal tract and enhance the vulnerability for invasive infection, thereby aggravating the severity of existing MOSF. Together with the predominance of Enterobacteriaceae in infected patients, these results suggest that translocation of intestinal bacteria and endotoxin may be important in the evolution and perpetuating the MOSF syndrome. Our results may be useful in devising strategies to prevent or limit the evolution of MOSF and to improve survival in patients with critical illness.