Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). However, the molecular mechanisms of NPC metastasis are poorly understood. Here, using our customized gene microarray containing all of the known human transcription factors and the current markers for epithelial-mesenchymal transition, we report that TEL2 was down-regulated in highly metastatic NPC cells and the metastatic tissues in lymph node. Mechanistically, TEL2 inhibits the cell migration and invasion in vitro and metastasis in vivo by directly suppressing the SERPINE1 promoter in NPC. Consistently, an inverse correlation was observed between the protein levels of TEL2 and SERPINE1 using clinical NPC samples. Collectively, we have provided the first evidence that TEL2 plays a key role in NPC metastasis by directly down-regulating SERPINE1, and that this novel axis of TEL2 / SERPINE1 may be valuable to develop new strategies for treating NPC patients with metastasis.

Dose assessment after radiological disasters is imperative to decrease mortality through rationally directed medical intervention. Our goal was to identify biomarkers capable of qualitative (nonirradiated/irradiated) and/or quantitative (dose) assessment of radiation exposure. Using real-time quantitative PCR, biodosimetry genes were identified in blood samples from cancer patients undergoing total-body irradiation. Time- (5, 12, 23, 48 h) and dose- (0-8 Gy) dependent changes in gene expression were examined in C57BL/6 mice. A training set was used to derive weighted voting classification algorithms (nonirradiated/irradiated) and continuous regression (dose assessment) models that were tested in a separate validation set of mice. Of eight biodosimetry genes identified in cancer patients ( ACTA2 , BBC3 , CCNG1 , CDKN1A , GADD45A , MDK , SERPINE1 , Tnfrsf10b ), expression of BBC3 , CCNG1 , CDKN1A , SERPINE1 and Tnfrsf10b was significantly (P < 0.05) increased in irradiated mice. CCNG1 and CDKN1A expression segregated irradiated mice from controls with an accuracy, specificity and sensitivity of 96.3, 100.0 and 94.4%, respectively, at 48 h. Multiple linear regression analysis predicted doses for the 0-, 1-, 2-, 4-, 6- and 8-Gy treatment groups as 0.0 ± 0.2, 1.6 ± 1.0, 2.9 ± 1.4, 5.1 ± 2.0, 5.3 ± 0.7 and 10.5 ± 5.6 Gy, respectively. These results suggest that gene expression analysis could be incorporated into biodosimetry protocols for qualitative and quantitative assessment of radiation exposure.

Hypertrophic cardiomyopathy (HCM) is the most common inherited primary myocardial disorder. HCM is characterized by interstitial fibrosis and excessive accumulation of extracellular matrix (ECM) proteins. Fibrosis in HCM has been associated with impaired cardiac function and heart failure, and has been considered a key substrate for ventricular arrhythmias and sudden death. The molecular triggers underpinning ECM production are not well established. We have previously developed a double-mutant mouse model of HCM that recapitulates the phenotype seen in humans with multiple mutations, including earlier onset of the disease, progression to a dilated phenotype, severe heart failure and premature mortality. The present study investigated the expression of ECM-encoding genes in severe HCM and heart failure. Significant upregulation of structural Fn1, regulatory Mmp14, Timp1, Serpin3A, SerpinE1, SerpineE2, Tgfβ1, and Tgfβ2; and matricellular Ccn2, Postn, Spp1, Thbs1, Thbs4, and Tnc was evident from the early, pre-phenotype stage. Non-myocytes expressed ECM genes at higher levels than cardiomyocytes in normal and diseased hearts. Synchronous increase of secreted CCN2 and TIMP1 plasma levels and decrease of MMP3 levels were observed in end-stage disease. CCN2 protein expression was increased from early disease in double-mutant hearts and played an important role in ECM responses. It was a powerful modulator of ECM regulatory (Timp1 and SerpinE1) and matricellular protein-encoding (Spp1, Thbs1, Thbs4 and Tnc) gene expression in cardiomyocytes when added exogenously in vitro. Modulation of CCN2 (CTGF, connective tissue growth factor) and associated early ECM changes may represent a new therapeutic target in the treatment and prevention of heart failure in HCM.

Patients with diabetes mellitus (DM) may develop corneal complications and delayed wound healing. The aims of this study are to characterize the molecular signatures and biological pathways leading to delayed epithelial wound healing and to delineate the involvement of TGFβ3 therein. Genome-wide cDNA microarray analysis revealed 1,888 differentially expressed genes in the healing epithelia of normal (NL) versus type 1 DM rat corneas. Gene ontology and enrichment analyses indicated TGFβ signaling as a major altered pathway. Among three TGFβ isoforms, TGF-β1 and β3 were upregulated in response to wounding in NL corneal epithelial cells (CECs), whereas the latter was greatly suppressed by hyperglycemia in rat type 1 and 2 and mouse type 1 DM models. Functional analysis indicated that TGF-β3 contributed to wound healing in NL corneas. Moreover, exogenously added TGF-β3 accelerated epithelial wound closure in type 2 rat and type 1 mouse DM corneas via Smad and PI3K-AKT signaling pathways, autoregulation, and/or upregulation of Serpine1, a well-known TGFβ target gene. Taken together, our study for the first time provides a comprehensive list of genes differentially expressed in the healing CECs of NL versus diabetic corneas and suggests the therapeutic potential of TGF-β3 for treating corneal and skin wounds in diabetic patients.

Intracranial aneurysm is probably a complex disease with both genetic and non-genetic or environmental risk factors contributing to the etiology of the disease. A disruption of the extracellular matrix (ECM) of the arterial wall is a likely factor in the pathogenesis of intracranial aneurysms. We analyzed 44 potential candidate genes involved in the maintenance of the integrity of the ECM in 382 Dutch Caucasian patients with intracranial aneurysms and 609 Dutch Caucasian controls for 384 tag single nucleotide polymorphisms (SNPs) using the GoldenGate assay on an Illumina BeadStation 500 GX. We identified SNPs that were associated with intracranial aneurysms (P<0.01) in six of these 44 genes: serpine1 (SERPINE1, P=0.0008), transforming growth factor beta induced (TGFBI, P=0.0026), perlecan (HSPG2, P=0.0044), fibronectin (FN1, P=0.0069), fibrillin 2 (FBN2, P=0.0077) and alpha 1 type IV collagen (COL4A1, P=0.0087). In a second independent cohort of 310 Dutch Caucasian intracranial aneurysm patients and 336 Dutch Caucasian controls, the association for the HSPG2 gene [combined odds ratio (OR) 1.33, 95% confidence interval (CI) 1.13-1.57, P=6 x 10(-4)] was replicated. The population attributable risk (PAR) for this SNP is 19%. Combining the two cohorts still showed association for the SERPINE1 (combined OR 1.27, 95% CI 1.07-1.50, P=0.004, PAR 6%), FBN2 (combined OR 1.37, 95% CI 1.07-1.75, P=0.01, PAR 3%) and COL4A1 (combined OR 1.22, 95% CI 1.05-1.42, P=0.007, PAR 7%) genes. These PARs are likely to be overestimates as they are calculated from the joint analyses combining stages 1 and 2 of our association study. Our findings indicate that variation in genes involved in the maintenance of the integrity of the ECM of the arterial wall plays a role in susceptibility to intracranial aneurysms. These findings further support our hypothesis that diminished maintenance of the ECM of the arterial wall is important in the development of intracranial aneurysms.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Wnt signaling plays an important role in preadipocyte differentiation and adipogenesis. The expression of a Wnt antagonist, secreted frizzled related protein 1 (SFRP1), is increased in response to initial weight gain, then levels are reduced under conditions of extreme obesity in both humans and animals. Here we report that loss of Sfrp1 exacerbates weight gain, glucose homeostasis and inflammation in mice in response to diet induced obesity (DIO). Sfrp1(-/-) mice fed a high fat diet (HFD) exhibited an increase in body mass accompanied by increases in body fat percentage, visceral white adipose tissue (WAT) mass, and adipocyte size. Moreover, Sfrp1 deficiency increases the mRNA levels of key de novo lipid synthesis genes (Fasn, Acaca, Acly, Elovl, Scd1) and the transcription factors that regulate their expression (Lxr-α, Srebp1, Chreb, and Nr1h3) in WAT. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated (G6pc and Pck1), and glucose transporters are repressed (Slc2a2 and Slc2a4) in Sfrp1(-/-) mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1(-/-) mice. When there is an expansion of adipose tissue there is a sustained inflammatory response accompanied by adipokine dysregulation, which leads to chronic subclinical inflammation. Thus, we assessed the inflammatory state of different tissues and revealed that Sfrp1(-/-) mice fed a HFD exhibited increased macrophage infiltration and expression of pro-inflammatory markers including IL-6, Nmnat, Tgf-β2, and SerpinE1. Our findings demonstrate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular signaling in response to the onset of obesity.

OBJECTIVE

Plasminogen activator inhibitor-1 (PAI-1, also known as serpin peptidase inhibitor, clade E [nexin, plasminogen activator inhibitor type 1], member 1 [SERPINE1]) plays a pathogenetic role in renal fibrosis. It is upregulated in experimental and human diabetic nephropathy. These studies assessed the effect of PAI-1 deficiency and overproduction on renal disease in experimental diabetes.

METHODS

Diabetes was induced by injection of streptozotocin in 6-week-old PAI-1-deficient mice, transgenic mice overexpressing Pai-1 and control mice. Animals were killed after 24 weeks of diabetes or after observation alone.

RESULTS

Pai-1 mRNA was upregulated in kidneys from genetically normal mice with diabetes and in non-diabetic Pai-1 transgenic mice. PAI-1 was not further increased in kidneys from Pai-1 transgenic mice with diabetes. Diabetes-associated albuminuria and glomerular injury, as well as renal alpha-smooth muscle actin production, were ameliorated in diabetic PAI-1-deficient mice, an amelioration associated with attenuated increases in renal matrix metallopeptidase-2 expression and activity. Diabetic Pai-1 transgenic mice did not develop increased albuminuria or glomerular injury, but the tubulointerstitial area was modestly enhanced. In addition to the findings in diabetic mice, abnormalities also developed in 30-week-old PAI-1-deficient and Pai-1 transgenic mice without diabetes. PAI-1 deficiency resulted in increased tubulointerstitial area, TGFB1 protein and alpha-smooth muscle actin. Non-diabetic 30-week-old Pai-1 transgenic mice developed similar renal abnormalities and increased matrix metallopeptidase-2 activity, together with a modest increase in serum glucose and HbA(1c).

CONCLUSIONS

These results demonstrate that endogenous PAI-1 deficiency protects mice from glomerular injury in longer term diabetes and that endogenous PAI-1 maintains normal renal interstitial structure in ageing not associated with diabetes.

High SERPINE1 expression is a common event in head and neck squamous cell carcinoma (HNSCC); however, whether it plays a role in determining clinical outcome remains still unknown. We studied SERPINE1 as a prognostic marker in two HNSCC patient cohorts. In a retrospective study (n = 80), high expression of SERPINE1 was associated with poor progression-free (p = 0.022) and cancer-specific (p = 0.040) survival. In a prospective study (n = 190), high SERPINE1 expression was associated with poor local recurrence-free (p = 0.022), progression-free (p = 0.002) and cancer-specific (p = 0.006) survival. SERPINE1 expression was identified as an independent risk factor for progression-free survival in patients treated with chemo-radiotherapy or radiotherapy (p = 0.043). In both patient cohorts, high SERPINE1 expression increased the risk of metastasis spread (p = 0.045; p = 0.029). The association between SERPINE1 expression and survival was confirmed using the HNSCC cohort included in The Cancer Genome Atlas project (n = 507). Once again, patients showing high expression had a poorer survival (p < 0.001). SERPINE1 over-expression in HNSCC cells reduced cell proliferation and enhanced migration. It also protected cells from cisplatin-induced apoptosis, which was accompanied by PI3K/AKT pathway activation. Downregulation of SERPINE1 expression had the opposite effect. We propose SERPINE1 expression as a prognostic marker that could be used to stratify HNSCC patients according to their risk of recurrence.

Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However, the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokine-related and inflammatory pathways. Remarkably, the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However, it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related, inflammatory pathways and self-renewal supporting gene, namely cyclin-encoding CCND2, and the associated genes CCNA1 and CCNE1. Conversely, we also see the down-regulation of the p53 (CCNB2, GTSE1, SERPINE1) and cell cycle (PLK1, CCNB2) pathways. Our data demonstrates that a cyclic AMP analog, 8-Br-cAMP, enhances the efficiency of cellular reprogramming. In addition, 8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming, which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming.

In the fetal lung, endogenous transforming growth factor (TGF)-beta inhibits early morphogenesis and blocks hormone-induced type II cell differentiation. We hypothesized that endogenous TGF-beta inhibits type II cell differentiation and that the stimulatory effects of glucocorticoids result in part from suppression of TGF-beta. Epithelial cells were isolated from human fetal lung and cultured under defined conditions with and without dexamethasone plus cAMP to promote type II cell differentiation. Control cells produced TGF-beta, which was activated in part by alpha(V)beta(6)-integrin. Treatment with dexamethasone, but not cAMP, reduced TGF-beta1 and -beta2 transcripts and TGF-beta bioactivity in culture medium. To examine the effects of decreased TGF-beta in the absence of glucocorticoid, cells were treated with antibodies to TGF-beta and its receptors. By real-time RT-PCR, antibody blockade of TGF-beta reduced serpine1, a TGF-beta-inducible gene, and increased gene expression for sftpa, sftpb, sftpc, and titf1, mimicking the response to hormone treatment. By microarray analysis, 29 additional genes were induced by both TGF-beta antibody and hormone treatment, and 20 other genes were repressed by both treatments. For some genes, the fold response was comparable for antibody and hormone treatment. We conclude that endogenous TGF-beta suppresses expression of surfactant proteins and selected other type II cell genes in fetal lung, in part secondary to increased expression of titf1, and we propose that the mechanism of glucocorticoid-induced type II cell differentiation includes antagonism of TGF-beta gene suppression. Surfactant production during fetal development is likely influenced by relative levels of TGF-beta and glucocorticoids.

BACKGROUND

Preeclampsia (PE) is a serious hypertensive pregnancy disorder with a significant genetic component. Numerous genetic studies, including our own, have yielded many susceptibility genes from distinct functional groups. Additionally, transcriptome profiling of tissues at the maternal-fetal interface has likewise yielded many differentially expressed genes. Often there is little overlap between these two approaches, although genes identified in both approaches are significantly associated with PE. We have thus taken a novel integrative bioinformatics approach of analysing pathways common to the susceptibility genes and the PE transcriptome.

METHODS

Using Illumina Human Ht12v4 and Wg6v3 BeadChips, transcriptome profiling was conducted on n = 65 normotensive and n = 60 PE decidua basalis tissues collected at delivery. The R software package libraries lumi and limma were used to preprocess transcript data for pathway analysis. Pathways were analysed and constructed using Pathway Studio. We examined ten candidate genes, which are from these functional groups: activin/inhibin signalling-ACVR1, ACVR1C, ACVR2A, INHA, INHBB; structural components-COL4A1, COL4A2 and M1 family aminopeptidases-ERAP1, ERAP2 and LNPEP.

CONCLUSIONS

Major common regulators/targets of these susceptibility genes identified were AGT, IFNG, IL6, INHBA, SERPINE1, TGFB1 and VEGFA. The top two categories of pathways associated with the susceptibility genes, which were significantly altered in the PE decidual transcriptome, were apoptosis and cell signaling (p < 0.001). Thus, susceptibility genes from distinct functional groups share similar downstream pathways through common regulators/targets, some of which are altered in PE. This study contributes to a better understanding of how susceptibility genes may interact in the development of PE. With this knowledge, more targeted functional analyses of PE susceptibility genes in these key pathways can be performed to examine their contributions to the pathogenesis and severity of PE.

Sinusoidal obstruction syndrome (SOS; formerly veno-occlusive disease) is a well-established complication of hematopoietic stem cell transplantation, pyrrolizidine alkaloid intoxication, and widely used chemotherapeutic agents such as oxaliplatin. It is associated with substantial morbidity and mortality. Pathogenesis of SOS in humans is poorly understood. To explore its molecular mechanisms, we used Affymetrix U133 Plus 2.0 microarrays to investigate the gene expression profile of 11 human livers with oxaliplatin-related SOS and compared it to 12 matched controls. Hierarchical clustering analysis showed that profiles from SOS and controls formed distinct clusters. To identify functional networks and gene ontologies, data were analyzed by the Ingenuity Pathway Analysis Tool. A total of 913 genes were differentially expressed in SOS: 613 being upregulated and 300 downregulated. Reverse transcriptase-PCR results showed excellent concordance with microarray data. Pathway analysis showed major gene upregulation in six pathways in SOS compared with controls: acute phase response (notably interleukin 6), coagulation system (Serpine1, THBD, and VWF), hepatic fibrosis/hepatic stellate cell activation (COL3a1, COL3a2, PDGF-A, TIMP1, and MMP2), and oxidative stress. Angiogenic factors (VEGF-C) and hypoxic factors (HIF1A) were upregulated. The most significant increase was seen in CCL20 mRNA. In conclusion, oxaliplatin-related SOS can be readily distinguished according to morphologic characteristics but also by a molecular signature. Global gene analysis provides new insights into mechanisms underlying chemotherapy-related hepatotoxicity in humans and potential targets relating to its diagnosis, prevention, and treatment. Activation of VEGF and coagulation (vWF) pathways could partially explain at a molecular level the clinical observations that bevacizumab and aspirin have a preventive effect in SOS.

BACKGROUND

Because of the faltering sensitivity and/or specificity, urine-based assays currently have a limited role in the management of patients with bladder cancer. The aim of this study was to externally validate our previously reported protein biomarker panel from multiple sites in the United States and Europe.

METHODS

This multicenter external validation study included a total of 320 subjects (bladder cancer = 183). The 10 biomarkers (IL8, MMP9, MMP10, SERPINA1, VEGFA, ANG, CA9, APOE, SDC1, and SERPINE1) were measured using commercial ELISA assays in an external laboratory. The diagnostic performance of the biomarker panel was assessed using receiver operator curves (ROC) and descriptive statistical values.

RESULTS

Utilizing the combination of all 10 biomarkers, the area under the ROC for the diagnostic panel was noted to be 0.847 (95% confidence interval, 0.796-0.899), outperforming any single biomarker. The multiplex assay at optimal cutoff value achieved an overall sensitivity of 0.79, specificity of 0.79, positive prediction value of 0.73, and negative prediction value of 0.84 for bladder cancer classification. Sensitivity values of the diagnostic panel for high-grade bladder cancer, low-grade bladder cancer, muscle invasive bladder cancer, and non-muscle invasive bladder cancer were 0.81, 0.90, 0.95, and 0.77, respectively.

CONCLUSIONS

Urinary levels of the biomarker panel enabled discrimination of patients with bladder cancer and controls, and the levels of biomarker subsets were associated with advancing tumor grade and stage.

CONCLUSIONS

If proven to be reliable, urinary diagnostic biomarker assays can detect bladder cancer in a timely manner such that the patient can expect improvements in overall survival and quality of life.

The zebrafish heart regenerates after ventricular damage through a process involving inflammation, fibrotic tissue deposition/removal and myocardial regeneration. Using 3D whole-mount imaging, we reveal a highly dynamic endocardium during cardiac regeneration, including changes in cell morphology, behaviour and gene expression. These events lay the foundation for an initial expansion of the endocardium that matures to form a coherent endocardial structure within the injury site. We studied two important endocardial molecules, Serpine1 and Notch, which are implicated in different aspects of endocardial regeneration. Notch signalling regulates developmental gene expression and features of endocardial maturation. Also, Notch manipulation interferes with attenuation of the inflammatory response and cardiomyocyte proliferation and dedifferentiation. serpine1 is strongly expressed very early in the wound endocardium, with decreasing expression at later time points. serpine1 expression persists in Notch-abrogated hearts, via what appears to be a conserved mechanism. Functional inhibition studies show that Serpine1 controls endocardial maturation and proliferation and cardiomyocyte proliferation. Thus, we describe a highly dynamic endocardium in the regenerating zebrafish heart, with two key endocardial players, Serpine1 and Notch signalling, regulating crucial regenerative processes.

Glioblastoma multiforme (GBM) displays high resistance to radiation and chemotherapy, due to the presence of a fraction of GBM stem-like cells (GSLCs), which are thus representing the target for GBM elimination. Since mesenchymal stem cells (MSCs) display high tumor tropism, we examined possible antitumor effects of the secreted factors from human MSCs on four GSLC lines (NCH421k, NCH644, NIB26, and NIB50). We found that conditioned media from bone marrow and umbilical cord-derived MSCs (MSC-CM) mediated cell cycle arrest of GSLCs by downregulating cyclin D1. PCR arrays revealed significantly deregulated expression of 13 genes associated with senescence in NCH421k cells exposed to MSC-CM. Among these, ATM, CD44, COL1A1, MORC3, NOX4, CDKN1A, IGFBP5, and SERPINE1 genes were upregulated, whereas IGFBP3, CDKN2A, CITED2, FN1, and PRKCD genes were found to be downregulated. Pathway analyses in GO and KEGG revealed their association with p53 signaling, which can trigger senescence via cell cycle inhibitors p21 or p16. For both, upregulated expression was proven in all four GSLC lines exhibiting senescence after MSC-CM exposure. Moreover, MSC paracrine signals were shown to increase the sensitivity of NCH421k and NCH644 cells toward temozolomide, possibly by altering them toward more differentiated cell types, as evidenced by vimentin and GFAP upregulation, and Sox-2 and Notch-1 downregulation. Our findings support the notion that MSCs posses an intrinsic ability to inhibit cell cycle and induce senescence and differentiation of GSLCs.

Vinblastine (VBL) is used to treat certain kinds of cancer including Hodgkin's lymphoma, lung cancer, breast cancer, testicular cancer and cervical carcinoma. However, the rapid development of resistance during therapy remains a major clinical challenge. In order to reverse cancer cell resistance, the goal of this study was to find differentially expressed genes and chromosomal alterations in multidrug resistant (MDR) KB-v1 cells, further to probe the relationship between drug resistance and differential genes, and chromosomal changes in MDR cancer cells. Comparative genomic hybridization (CGH) analysis of MDR KB-v1 and their parental KB-3-1 cells revealed chromosomal changes; microarray-based expression profiling was carried out by comparing the gene expressions of MDR KB-v1 cells and KB-3-1 cells. We have identified 3 chromosomal gains in regions of 1p31, 7q21 and 18q21 in MDR cells and 10 genes (CYR61, UGTREL7, MBD1, NARS, ATP5A1, ABCB1, ABCB4, PEG10, MCM7, SERPINE1) contained in these regions were also up-regulated in MDR KB-v1 cells. Forty-nine genes were down-regulated when KB-v1 cells were subjected to lower dose or depletion of the drug. We have confirmed some gene expression changes by reverse transcription-polymerase chain reaction and Northern blots. These are the first data describing the relationship of 1p31 and 18q21 chromosomal aberrations and candidate genes in acquired vinblastine-resistance. This study also demonstrates that the combination of CGH and cDNA microarray is a very useful tool to detect drug resistant targets in cancer treatment.

OBJECTIVE

Evidence suggests that plasminogen activator inhibitor type 1 gene (SERPINE1) is a stress-related gene and serum plasminogen activator inhibitor type 1 levels are increased in patients with major depressive disorders (MDD).

METHODS

To investigate whether common genetic variation in the SERPINE1 gene is associated with MDD and the therapeutic response to antidepressants, six polymorphisms (rs2227631, rs1799889, rs6092, rs6090, rs2227684 and rs7242) of the SERPINE1 gene were genotyped in 188 Chinese MDD patients and 346 controls. Among the MDD patients, 140 accepted selective serotonin reuptake inhibitor (fluoxetine or citalopram) antidepressant treatment for 4 weeks with therapeutic evaluation before and after.

RESULTS

In single-marker-based analysis, the rs2227684-G and rs7242-T alleles were more frequent in MDD patients than in controls (P=0.010 and 0.010, respectively). The haplotype derived from the rs6090-G, rs2227684-G and rs7242-T polymorphisms was 1.19-fold higher in patients with MDD than in controls (P=0.0038). Haplotype analysis in responders (defined as a 50% reduction of the initial Hamilton score) and nonresponders after 4 weeks of antidepressant treatment showed that the haplotype derived from the rs2227631-G and rs1799889-4G polymorphisms was lower in responders than nonresponders (11.4 vs. 22.4%, P=0.014).

CONCLUSIONS

Our findings show, for the first time, that SERPINE1 genetic variants may play a role in MDD susceptibility and in the acute therapeutic response to selective serotonin reuptake inhibitors.

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β-mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1-dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

OBJECTIVE

To evaluate the expression of atherosclerosis-associated genes in patients with hypertension and type 2 diabetes mellitus.

METHODS

Twenty-seven patients (14 men, 13 women), mean age 43.26 ± 11.69 years, were included in the study, which was divided into three groups: group 1 - patients with newly diagnosed hypertension and normal glucose tolerance (n = 9), group 2 - normotensive individuals with newly diagnosed type 2 diabetes (n = 9), and control group - normotensive individuals with normal glucose tolerance (n = 9). Gene expression analysis was performed with Human Atherosclerosis RT2 Profiler PCR Array.

RESULTS

In patients with hypertension, we found eight genes with increased expression - FABP3, FAS, FN1, IL1R2, LPL, SERPINE1, TGFB1, and VCAM1. Decreased expression was observed for two genes - SELPLG and SERPINEB2. In patients with type 2 diabetes we found seven up-regulated genes - APOE, BAX, MMP1, NFKB1, PDGFB, SPP1, and TGFB2, whereas no specifically down-regulated genes were observed. Three genes - KLF2, PDGFRB, and PPARD were found to be expressed only in groups 1 and 2.

CONCLUSIONS

Hypertension is associated with increased expression of FABP3, FAS, FN1, IL1R2, LPL, SERPINE1, TGFB1, and VCAM1 and decreased expression of SELPLG and SERPINEB2. The up-regulation of FAS, FN1, SERPINE1, TGFB1, and VCAM1 might be associated with an increased cardiovascular risk. Type 2 diabetes is associated with increased expression of APOE, BAX, MMP1, NFKB1, PDGFB, SPP1, and TGFB2. KLF2 and PPARD might be part of protective mechanisms that limit target organ damage in both disease conditions. Expression of PDGFRB might play an important role in the pathogenesis of both hypertension and type 2 diabetes.

Progress in our understanding of thymic epithelial cell (TEC) renewal and homeostasis is hindered by the lack of markers for TEC progenitors. Stem and progenitor cell populations display remarkable diversity in their proliferative behavior. In some but not all tissues, stemness is associated with quiescence. The primary goal of our study was to discover whether quiescent cells were present in neonatal and adult TECs. To this end, we used a transgenic label-retaining cell (LRC) assay in which a histone H2B-GFP fusion protein is expressed under the control of the reverse tetracycline-controlled transactivator and the tetracycline operator minimal promoter. In adult mice, we found that both cortical and medullary TECs (cTECs and mTECs) proliferated more actively in females than males. Moreover, we observed three main differences between neonatal and adult TECs: 1) neonatal TECs proliferated more actively than adult TECs; 2) whereas cTECs and mTECs had similar turnover rates in young mice, the turnover of mTECs was more rapid than that of cTECs in adults; and 3) although no LRCs could be detected in young mice, LRCs were detectable after a 16-wk chase in adults. In female mice, LRCs were found almost exclusively among cTECs and expressed relatively low levels of p16INK4a, p19ARF, and Serpine1, and high levels of Bmi1, Foxn1, Trp63, and Wnt4. We conclude that LRCs in adult TECs are not senescent postmitotic cells and may represent the elusive progenitors responsible for TEC maintenance in the adult thymus.

The hypothesis that ovine luteal gene expression differs due to pregnancy status and day of estrous cycle was tested. RNA was isolated from corpora lutea (CL) on days 12 and 14 of the estrous cycle (NP) or pregnancy (P) and analyzed with the Affymetrix bovine microarray. RNA also was isolated from luteal cells on day 10 of estrous cycle that were cultured for 24 h with luteolytic hormones (OXT and PGF) and secretory products of the conceptus (IFNT and PGE2). Differential gene expression (>1.5-fold, P < 0.05) was confirmed using semiquantitative real-time PCR. Serum progesterone concentrations decreased from day 12 to day 15 in NP ewes (P < 0.05) reflecting luteolysis and remained >1.7 ng/ml in P ewes reflecting rescue of the CL. Early luteolysis (days 12-14) was associated with differential expression of 683 genes in the CL, including upregulation of SERPINE1 and THBS1. Pregnancy on day 12 (55 genes) and 14 (734 genes) also was associated with differential expression of genes in the CL, many of which were ISGs (i.e., ISG15, MX1) that were induced when culturing luteal cells with IFNT, but not PGE2. Finally, many genes, such as PTX3, IL6, VEGF, and LHR, were stabilized during pregnancy and downregulated during the estrous cycle and in response to culture of luteal cells with luteolytic hormones. In conclusion, pregnancy circumvents luteolytic pathways and activates or stabilizes genes associated with interferon, chemokine, cell adhesion, cytoskeletal, and angiogenic pathways in the CL.

Gastric adenocarcinoma (GAC), also known as stomach adenocarcinoma (STAD), is one of the most lethal malignancies in the world. It is vital to classify and detect the hub genes and key pathways participated in the initiation and progression of GAC. In this study, we collected and sequenced 15 pairs of GAC tumor tissues and the adjacent normal tissues. Differentially expressed genes (DEGs) were analyzed and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis were used to annotate the unique biological significance and important pathways of enriched DEGs. Moreover, we constructed the protein-protein interaction (PPI) network by Cytoscape and conducted KEGG enrichment analysis of the prime module. We further applied the TCGA database to start the survival analysis of these hub genes by Kaplan-Meier estimates. Finally, we obtained total 233 DEGs consisted of 64 up-regulated genes and 169 down-regulated genes. GO enrichment analysis found that DEGs most significantly enriched in single organism process, extracellular region, and extracellular region part. KEGG pathway enrichment analysis suggested that DEGs most significantly enriched in Protein digestion and absorption, Gastric acid secretion, and ECM-receptor interaction. Furthermore, the PPI network showed that the top 10 hub genes in GAC were IL8, COL1A1, MMP9, SST, COL1A2, TIMP1, FN1, SPARC, ALDH1A1, and SERPINE1 respectively. The prime gene interaction module in PPI network was enriched in protein digestion and absorption, ECM receptor interaction, the PI3K-Akt signaling pathway, and pathway in cancer. Survival analysis based on the TCGA database found that the expression of the FN1, SERPINE1, and SPARC significantly predicted poor prognosis of GAC. Collectively, we identified several hub genes and key pathways associated with GAC initiation and progression by analyzing the microarray data on DEGs, which provided a detailed molecular mechanism underlying GAC occurrence and progression.