Fundamentally, microglia have two activation states, a pro-inflammatory neurotoxic (M1) and an anti-inflammatory neuroprotective (M2) phenotype, and their conversion from M1-like to M2-like microglia may provide therapeutic benefits to prevent neuronal loss in neurodegenerative diseases such as Parkinson's disease (PD). Previously, we showed that Salmeterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, has neuroprotective effects in PD models in vitro and in vivo through the β-arrestin2-dependent inhibition of pro-inflammatory M1-type mediator production. In the present study, we explored whether Salmeterol can mediate phenotypic conversion in LPS-activated murine microglial BV2 cells from the neurotoxic M1-like to a neuroprotective M2-like phenotype. Salmeterol inhibited the production of LPS-induced mediators of the pro-inflammatory M1 phenotype such as tumor necrosis factor-α (TNF-α), IL-(interleukin) 18, IL-6, chemokines (CCL2, CCL3, CCL4) and reactive oxygen species from BV2 cells. Conversely, treatment with Salmeterol and other β2-AR agonists robustly enhances the production of the M2 cytokine IL-10 from LPS-activated microglia. In addition, Salmeterol upregulates the expression of arginase-1 and CXCL14. Furthermore, using siRNA approach we found that silencing of the transcription factor Creb abrogates the Salmeterol-mediated production of IL-10 in LPS-activated BV2 cells, but silencing of β-arrestin2 with Arrb2 siRNA did not. In addition, our data shows conversion from an M1- to M2-like phenotype in LPS-activated microglia by β2-AR agonists involves activation of the classical cAMP/PKA/CREB as well as the PI3K and p38 MAPK signaling pathways, and provides a novel therapeutic approach targeting microglial cell activation and inducing their phenotypic conversion in the treatment of neuroinflammatory diseases such as PD.

Gastrointestinal stromal tumors (GIST) are the most prevalent mesenchymal tumors of the digestive tract. To investigate the association of imatinib mesylate plasma concentration with adverse drug reactions (ADRs) and influences of genetic polymorphisms on ADRs in GIST patients taking imatinib, a cohort of GIST patients consecutively treated with imatinib were included in the observational study. Clinical, pathologic and genotype information was recorded at enrollment and blood samples were collected at time as design. The plasma concentration of the imatinib was detected by LC-MS/MS. A questionnaire was used to evaluate the ADRs at each visit. SNPs in 13 genes were analyzed for a possible association with ADRs. The mean plasma trough concentration of 129 patients taking imatinib was 1.45 ± 0.79 μg/ml, average peak concentration was 2.63 ± 1.07 μg/ml. The imatinib concentration in patients treated with 600 mg/day was significantly higher than other dosage groups (P < 0.05). The ADRs were mostly mild. Edema, vomiting, and fatigue were significantly correlated with imatinib concentration (P < 0.05). Mutations of IL13 rs1800925 and CXCL14 rs7716492 were related with the incidence of leukopenia and rash in our research, separately (P < 0.05). We confirmed that with the increase of imatinib concentration, the incidence of edema, vomiting, and fatigue rises as well. Mutations of IL13 rs1800925 and CXCL14 rs7716492 may be the promising biomarkers to predict the ADRs of imatinib. The results of the study are of guiding significance for the use of imatinib in patients with GIST.

Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor of the upper aerodigestive tract. The loss and gain of miRNA function promote cancer development through various mechanisms. RNA sequencing (RNA-seq) and miRNAs sequencing data from the Cancer Genome Atlas (TCGA) was used to show the dysfunctional miRNAs microenvironment and to provide useful biomarkers for miRNAs therapy. Seven miRNAs were found to be independent prognostic factors of HNSCC patients in the training cohort. A total of 60 target genes for these miRNAs were predicted. Nine target genes (CDCA4, CXCL14, FLNC, KLF7, NBEAL2, P4HA1, PFKM, PFN2 and SEPPINE1) were correlated with patient's overall survival (OS) outcomes. We identified novel miRNAs markers for the prognosis of head and neck squamous cell carcinoma.

Keywords: Head and neck squamous cell carcinoma; Prognosis; TCGA; miRNAs.

Gastrointestinal stromal tumors (GISTs) are defined as spindle cell and/or epithelioid tumors originated from interstitial Cajal cells or precursors in the digestive tract. This study was conducted to identify genes differing in expression between the gastric tumors and the adjacent non-cancerous mucosas in patients with primary gastric GIST. The gene expression profile was determined by using oligonucleotide-based DNA microarrays and further validated by quantitative real-time PCR. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed to predict signaling pathways involved in gastric GIST. Our data showed that the expression levels of 957 genes (RAB39B, member RAS oncogene family; VCAN, versican; etc.) were higher and that of 526 genes (CXCL14, chemokine C-X-C motif ligand 14; MTUS1, microtubule-associated tumor suppressor 1; etc.) were lower in the gastric tumor tissues as compared with normal gastric tissues. Results from KEGG pathway analysis revealed that the differentially expressed genes were enriched into 16 signaling transduction pathways, including Hedeghog and Wnt signaling pathways. Our study may provide basis for identification of novel biomarkers associated with primary gastric GIST pathogenesis and for exploration of underlying mechanisms involved in this gastric sarcoma.

In order to find a suppressor(s) of tumor progression in vivo for head and neck squamous cell carcinoma (HNSCC), we searched for molecules downregulated in HNSCC cells when the cells were treated with epidermal growth factor (EGF), whose receptor is frequently overactivated in HNSCC. The expression of BRAK, which is also known as CXC chemokine ligand 14 (CXCL14), was downregulated significantly by the treatment of HNSCC cells with EGF as observed by cDNA microarray analysis followed by reverse-transcriptase polymerase chain reaction analysis and western blotting. The EGF effect on the expression of CXCL14/BRAK was attenuated by the copresence of inhibitors of the EGF receptor, MEK, and ERK. The rate of tumor formation in vivo of BRAK-expressing vector-transfected tumor cells in athymic nude mice or SCID mice was significantly lower than that of mock vector-transfected ones. In addition tumors formed in vivo by the BRAK-expressing cells were significantly smaller than those of the mock-transfected ones. These results indicate that CXCL14/BRAK is a chemokine having suppressive activity toward tumor progression of HNSCC in vivo. Our approach will be useful to find new target molecules to suppress progression of tumors of various origins in addition to HNSCC.

The rumen immune system often suffers when challenging antigens from lysis of dead microbiota cells in the rumen. However, the rumen epithelium innate immune system can actively respond to the infection. Previous studies have demonstrated G protein-coupled receptors 41 (GPR41) as receptors for short chain fatty acids (SCFAs) in human. We hypothesized that SCFAs, the most abundant microbial metabolites in rumen, may regulate the immune responses by GPR41 in bovine rumen epithelial cells (BRECs). Therefore, the objective of study was to firstly establish an immortal BRECs line and investigate the regulatory effects of SCFAs and GPR41 on innate immunity responses in BRECs. These results showed that long-term BRECs cultures were established by SV40T-induced immortalization. The concentrations of 20 mM SCFAs significantly enhanced the levels of GPR41, IL1β, TNFα, chemokines, and immune barrier genes by transcriptome analysis. Consistent with transcriptome results, the expression of GPR41, IL1β, TNFα, and chemokines were markedly upregulated in BRECs treated with 20 mM SCFAs by qRT-PCR compared with control BRECs. Remarkably, the GPR41 knockdown (GPR41KD) BRECs treated with 20 mM SCFAs significantly enhanced the proinflammatory cytokines IL1β and TNFα expression compared with wild type BRECs treated with 20 mM SCFAs, but reduced the expression of CCL20, CXCL2, CXCL3, CXCL5, CXCL8, CXCL14, Occludin, and ZO-1. Moreover, GPR41 mRNA expression is positively correlated with CCL20, CXCL2, CXCL3, CXCL8, CXCL14, and ZO-1. These findings revealed that SCFAs regulate GPR41-mediated levels of genes involved in immune cell recruitment and epithelial immune barrier and thereby mediate protective innate immunity in BRECs.

BACKGROUND

Ingenol mebutat (IM)-gel is effective for the topical treatment of epithelial tumors, including actinic keratoses (AKs) or anogenital warts (AGW). AK patients treated with IM develop intensified inflammatory reactions on sights of prior clinical visible or palpable AKs as compared to the surrounding actinically damaged skin, suggesting the induction of a tumor cell-directed inflammation. AGW patients treated with IM develop even stronger inflammatory reactions with large erosions, suggesting a directed inflammatory response against HPV-infected keratinocytes. Of note, even widespread erosions heal very fast without any superinfections. Here, we set out to elucidate underlying molecular and cellular mechanisms of these clinical observations.

METHODS

The effects of IM (10-9-10-5 M) on the expression and translation of a comprehensive set of chemokines (CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCL14, CCL2, CCL5, CCL20, CCL27) and antimicrobial peptides (AMP) (HBD1, HBD2, HBD3, LL37, RNase7) were analyzed in primary human epithelial keratinocytes (HEK) and a set of epithelial cancer cell lines by RT-qPCR and ELISA in vitro. To study the possible effects of different concentrations of IM on migratory, respectively wound healing responses, an in vitro scratch assay was conducted on HEK.

RESULTS

Ingenol mebutat significantly and dose-dependently induced the expression of proinflammatory chemokines (CXCL8, CCL2) and AMP (RNase7, HBD3) in HEK and epithelial cancer cell lines. A significantly stronger induction of CXCL8 and CCL2 was observed in our tested tumor cells as compared to HEK. We did not observe any significant effect of IM on HEK migration, respectively wound healing responses in vitro for any tested concentration (10-9, 10-8, 10-6 M) except 10-7 M, which induced a significant inhibition.

CONCLUSIONS

Our data suggest that tumor cells are more susceptible to IM as compared to differentiated HEK. This is evident by a stronger IM-mediated induction of proinflammatory chemokines in tumor cells, which may result in a tumor cell-directed inflammatory response and rapid tumor destruction. In addition, IM induces AMP in keratinocytes and seems not to severely interfere with keratinocyte migration, which contributes to a fast and uncomplicated wound healing. Surprising is a selective inhibition of keratinocyte migration by IM at the concentration of 10-7 M pointing to very dose depending biological effects, induced by IM.

Evasion of the host immune responses is critical for both persistent human papillomavirus (HPV) infection and associated cancer progression. We have previously shown that expression of the homeostatic chemokine CXCL14 is significantly downregulated by the HPV oncoprotein E7 during cancer progression. Restoration of CXCL14 expression in HPV-positive head and neck cancer (HNC) cells dramatically suppresses tumor growth and increases survival through an immune-dependent mechanism in mice. Although CXCL14 recruits natural killer (NK) and T cells to the tumor microenvironment, the mechanism by which CXCL14 mediates tumor suppression through NK and/or T cells remained undefined. Here we report that CD8⁺ T cells are required for CXCL14-mediated tumor suppression. Using a CD8⁺ T-cell receptor transgenic model, we show that the CXCL14-mediated antitumor CD8⁺ T-cell responses require antigen specificity. Interestingly, CXCL14 expression restores major histocompatibility complex class I (MHC-I) expression on HPV-positive HNC cells downregulated by HPV, and knockdown of MHC-I expression in HNC cells results in loss of tumor suppression even with CXCL14 expression. These results suggest that CXCL14 enacts antitumor immunity through restoration of MHC-I expression on tumor cells and promoting antigen-specific CD8⁺ T-cell responses to suppress HPV-positive HNC.

Hematopoietic cell transplantation (HCT) is the only cure for sickle cell disease (SCD), but engraftment remains challenging in patients lacking matched donors. Infusion of mesenchymal stromal cells (MSCs) at the time of HCT may promote hematopoiesis and ameliorate graft-versus-host disease. Experimental murine models suggest MSC major histocompatibility complex compatibility with recipient impacts their in vivo function, suggesting autologous MSCs could be superior to third-party MSCs for promoting HCT engraftment. Here we tested whether bone marrow (BM)-derived MSCs from SCD subjects have comparable functionality compared with MSCs from healthy volunteers. SCD MSC doubling time and surface marker phenotype did not differ significantly from non-SCD. Third-party and autologous (SCD) T cell proliferation was suppressed in a dose-dependent manner by all MSCs. SCD MSCs comparably expressed indoleamine-2,3-dioxygenase, which based on transwell and blocking experiments appeared to be the dominant immunomodulatory pathway. The expression of key genes involved in hematopoietic stem cell (HSC)-MSC interactions was minimally altered between SCD and non-SCD MSCs. Expression was, however, altered by IFN-γ stimulation, particularly CXCL14, CXCL26, CX3CL1, CKITL, and JAG1, indicating the potential to augment MSC expression by cytokine stimulation. These data demonstrate the feasibility of expanding BM-derived MSCs from SCD patients that phenotypically and functionally do not differ per International Society of Cell Therapy essential criteria from non-SCD MSCs, supporting initial evaluation (primarily for safety) of autologous MSCs to enhance haploidentical HSC engraftment in SCD.

Peroxisome proliferator-activated receptor α/δ (PPAR α/δ), regulating glucolipid metabolism and immune inflammation, has been identified as an effective therapeutic target in non-alcoholic steatohepatitis (NASH). Dual PPAR α/δ agonist, such as GFT505 (also known as elafibranor), demonstrated potential therapeutic effect for NASH in clinical trials. To profile the regulatory network of PPAR α/δ agonist in NASH, the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) induced NASH model was used to test the pharmacodynamics and transcriptome regulation of GFT505 in this study. The results showed that GFT505 ameliorated hepatic steatosis, inflammation and fibrosis in CDAHFD mice model. RNA-sequencing yielded 3995 up-regulated and 3576 down-regulated genes with GFT505 treatment. And the most significant differentialy expressed genes involved in glucolipid metabolism (Pparα, Acox1, Cpt1b, Fabp4, Ehhadh, Fabp3), inflammation (Ccl6, Ccl9, Cxcl14) and fibrosis (Timp1, Lamc3, Timp2, Col3a1, Col1a2, Col1a1, Hapln4, Timp3, Pik3r5, Pdgfα, Pdgfβ, Tgfβ1, Tgfβ2) were confirmed by RT-qPCR. The down-regulated genes were enriched in cytokine-cytokine receptor interaction pathway and ECM-receptor interaction pathway, while the up-regulated genes were enriched in PPAR signaling pathway and fatty acid degradation pathway. This study provides clues and basis for further understanding on the mechanism of PPAR α/δ agonist on NASH.

Cancer is a multifactorial disease driven by a combination of genetic and environmental factors. Many cancer driver mutations have been characterised in protein-coding regions of the genome. However, mutations in noncoding regions associated with cancer have been less investigated. G-quadruplex (G4) nucleic acids are four-stranded secondary structures formed in guanine-rich sequences and prevalent in the regulatory regions. In this study, we used published whole cancer genome sequence data to find mutations in cancer patients that overlap potential RNA G4-forming sequences in 5' UTRs. Using RNAfold, we assessed the effect of these mutations on the thermodynamic stability of predicted RNA G4s in the context of full-length 5' UTRs. Of the 217 identified mutations, we found that 33 are predicted to destabilise and 21 predicted to stabilise potential RNA G4s. We experimentally validated the effect of destabilising mutations in the 5' UTRs of BCL2 and CXCL14 and one stabilising mutation in the 5' UTR of TAOK2. These mutations resulted in an increase or a decrease in translation of these mRNAs, respectively. These findings suggest that mutations that modulate the G4 stability in the noncoding regions could act as cancer driver mutations, which present an opportunity for early cancer diagnosis using individual sequencing information.

Glioblastoma (GBM) is a primary brain tumor whose prognosis is inevitably dismal, leading patients to death in about 15 months from diagnosis. Tumor cells in the mass of the neoplasm are in continuous exchange with cells of the stromal microenvironment, through the production of soluble molecules, among which chemokines play prominent roles. CXCL14 is a chemokine with a pro-tumor role in breast and prostate carcinoma, where it is secreted by cancer associated fibroblasts, and contributes to tumor growth and invasion. We previously observed that CXCL14 expression is higher in GBM tissues than in healthy white matter. Here, we study the effects of exogenously supplemented CXCL14 on key tumorigenic properties of human GBM cell lines. We show that CXCL14 enhances the migration ability and the proliferation of U87MG and LN229 GBM cell lines. None of these effects was affected by the use of AMD3100, an inhibitor of CXCR4 receptor, suggesting that the observed CXCL14 effects are not mediated by this receptor. We also provide evidence that CXCL14 enhances the sphere-forming ability of glioblastoma stem cells, considered the initiating cells, and is responsible for tumor onset, growth and recurrence. In support of our in vitro results, we present data from several GBM expression datasets, demonstrating that CXCL14 expression is inversely correlated with overall survival, that it is enriched at the leading edge of the tumors and in infiltrating tumor areas, and it characterizes mesenchymal and NON G-CIMP tumors, known to have a particularly bad prognosis. Overall, our results point to CXCL14 as a protumorigenic chemokine in GBM.

OBJECTIVE

The chemokines CXCL10 (interferon ϒ-inducible protein 10 [IP-10]), CXCL11 (Human interferon inducible T cell alpha chemokine [I-TAC]), CXCL12 (stromal cell derived factor 1 [SDF-1]), and CXCL14 (breast and kidney-expressed chemokine [BRAK]) are involved in cell recruitment, migration, activation, and homing in liver diseases and have been shown to be upregulated during acute liver injury in animal models. However, their expression in patients with acute liver injury is unknown. Here, we aimed to provide evidence of the presence of circulating CXCL10, CXCL11, CXCL12, and CXCL14 during human acute liver injury to propose new inflammation biomarkers for acute liver injury.

METHODS

We analyzed the serum concentration of the studied chemokines in healthy donors (n = 36) and patients (n = 163) with acute liver injuries of various etiologies.

RESULTS

Serum CXCL10, CXCL11 and CXCL12 levels were elevated in all the studied groups except biliary diseases for CXCL11. CXCL14 was associated with only acute viral infection and vascular etiologies. The strongest correlation was found between the IFN-inducible studied chemokines (CXCL10 and CXCL11) in all patients and more specifically in the acute viral infection group.

CONCLUSIONS

These data provide evidence for the presence of circulating CXCL10, CXCL11, CXCL12, and CXCL14 during acute liver injury and are consistent with data obtained in animal models. CXCL10, CXCL11 and CXCL12 were the most highly represented and CXCL14 the least represented chemokines. Differential expression patterns were obtained depending on acute liver injury etiology, suggesting the potential use of these chemokines as acute liver injury biomarkers.

Circular RNAs (circRNAs) function as efficient microRNA (miRNA) sponges that regulate gene expression in the pathogenesis of many human malignancies. However, their roles in cervical adenocarcinoma remain largely unknown. In this study, we aimed to seek novel circRNAs that regulate cervical adenocarcinoma carcinogenesis and to explore their regulatory mechanisms as well as clinical significance. We identified that 24 circRNAs were differentially expressed in cervical adenocarcinoma tissues by RNA sequencing. Among them, circEYA1 was the most significantly downregulated circRNA in cervical adenocarcinoma. In cervical adenocarcinoma cells, circEYA1 overexpression led to suppression of cell viability and colony formation, promotion of apoptosis, and a decrease of the xenograft tumor growth. The mechanism underlying these observations is that circEYA1 functioned as a sponge of miR-582-3p and abrogated its suppression of CXCL14 expression. Consistently, miR-582-3p inhibition phenocopied the biological effects of circEYA1 overexpression in cervical adenocarcinoma cells. Moreover, miR-582-3p overexpression reversed the suppressive behaviors of circEYA1 in vitro and in vivo. In addition, the expression, correlation, and clinical diagnostic value of circEYA1/miR-582-3p/CXCL14 were confirmed in 198 clinical cervical tissue samples. In summary, our findings highlight a novel tumor suppressive role of circEYA1 in cervical adenocarcinoma tumorigenesis and may provide a potential diagnostic marker and therapeutic target for patients with cervical adenocarcinoma.

Keywords: CXCL14; cell viability; cervical adenocarcinoma; circEYA1; miR-582-3p.

Background: The presence of neutrophil-rich inflammation in colon tissues of patients with ulcerative colitis (UC) is one of the most important histological characteristics of this disease. However, the expression of CXCL chemokines governing the infiltration of neutrophils in UC has not been well elucidated. Materials and methods: In this experimental study, the UC model was induced in Wistar rats by administration of 2 mL 4% acetic acid into the large colon through the rectum. Animals were anesthetized after 48 hrs; their colon tissue samples were isolated for macroscopic and histopathological examinations. The expression of CXCL family was assessed by reverse transcription polymerase chain reaction (qRT-PCR) technique. Results: Heavy infiltration of neutrophils, coagulation necrosis, and ulcers were observed in H&E staining, which pathologically proved the UC model. qRT-PCR results showed that ELR⁺ CXC chemokines such as CXCL6 and CXCL3 had the highest expression in the UC group, which was 49 and 28 times higher than that of the control group, respectively. In addition, other chemokines of this group including CXCL1, CXCL2, and CXCL7 had a significant increase compared to the control group (P≤0.05). However, ELR^- CXC chemokines such as CXCL4, CXCL13, and CXCL16 showed a smaller upregulation, while CXCL14 chemokine showed a significant decrease compared to the control group (P≤0.05). However, the expression of CXCL9-12 and CXCL17 did not change. Conclusion: The results showed that the ELR⁺ CXC chemokines, especially CXCL6 and CXCL3, many involved in the pathogenesis of UC; therefore, CXCL6 and CXCL3 chemokines can be used as therapeutic targets for UC, although more studies using human samples are required.

The aim of the study was to explore the expression of three genes, HSPD1, SCUBE3, and CXCL14, in osteosarcoma cells and tissue, as well as their association with the prognosis of patients with osteosarcoma. The expression of HSPD1, SCUBE3, and CXCL14 in osteosarcoma cells was detected by using Western blotting method. siRNA was used to knockdown the expression of the three genes. CCK8 cell proliferation assay was used to observe the effect of siRNA interference on U2OS cell proliferation. The expression of the three genes in osteosarcoma tissue was detected employing immunohistochemical method. Kaplan-Meier survival analysis was used to compare the relations between the expression of the three genes and prognosis. The Western blotting results showed that the expression of Hsp70, SCUBE3 protein, and CXCL14 chemotactic factor in osteosarcoma cells was significantly higher than that in normal osteocytes (p < 0.05). After the three genes were interfered by siRNA, the mRNA and protein expression levels of these genes in osteosarcoma cells were significantly decreased (p < 0.05). The growth rate of U2OS cell after the siRNA interference was significantly lower than that before interference and that in the control group transfected with negative control siRNA (p < 0.05). The result of immunohistochemistry demonstrated that the expression of Hsp70, SCUBE3 protein, and CXCL14 chemotactic factor in osteosarcoma tissues was significantly higher than that in adjacent muscle tissue (p < 0.05). Kaplan-Meier survival analysis indicated that the survival rate of the patients with high expression of those three kinds of genes was obviously lower than that of other patients (p < 0.05). There was no significant difference between the survival rates of patients with high or low expression of two genes (p>> 0.05). The expression of HSPD1, SCUBE3, and CXCL14 was all high in osteosarcoma tissues and cells; moreover, the three kinds of genes had close correlations with the prognosis of the patients. Targeted inhibition of these three genes could inhibit the proliferation of the tumor, which may become a new therapeutic target.

BACKGROUND

Random periareolar fine-needle aspiration (RP-FNA) is increasingly used in trials of breast cancer prevention for biomarker assessments. DNA methylation markers may have value as surrogate endpoint biomarkers, but this requires identification of biologically relevant markers suitable for paucicellular, lymphocyte-contaminated clinical samples.

METHODS

Unbiased whole-genome 5-aza-2'-deoxycytidine (5AZA)-induced gene expression assays, followed by several phases of qualitative and quantitative methylation-specific PCR (MSP) testing, were used to identify novel breast cancer DNA methylation markers optimized for clinical FNA samples.

RESULTS

The initial 5AZA experiment identified 453 genes whose expression was potentially regulated by promoter region methylation. Informatics filters excluded 273 genes unlikely to yield useful DNA methylation markers. MSP assays were designed for 271 of the remaining genes and, ultimately, 33 genes were identified that were differentially methylated in clinical breast cancer samples, as compared with benign RP-FNA samples, and never methylated in lymphocytes. A subset of these markers was validated by quantitative multiplex MSP in extended clinical sample sets. Using a novel permutation method for analysis of quantitative methylation data, PSAT1, GNE, CPNE8, and CXCL14 were found to correlate strongly with specific clinical and pathologic features of breast cancer. In general, our approach identified markers methylated in a smaller subpopulation of tumor cells than those identified in published methylation array studies.

CONCLUSIONS

Clinically relevant DNA methylation markers were identified using a 5AZA-induced gene expression approach.

CONCLUSIONS

These breast cancer-relevant, FNA-optimized DNA methylation markers may have value as surrogate endpoint biomarkers in RP-FNA studies.

OBJECTIVE

A low pH microenvironment is a characteristic feature of inflammation loci and affects the functions of immune cells. In this study, we investigated the effect of extracellular acidification on macrophage gene expression.

METHODS

RAW264.7 macrophages were incubated in neutral (pH 7.4) or acidic (pH 6.8) medium for 4 h. Global mRNA expression levels were determined using Affymetrix genechips.

RESULTS

The mRNA expressions of 353 macrophage genes were significantly modified after incubation in acidic medium; 193 were up-regulated and 160 down-regulated. Differentially regulated genes were grouped into 13 classes based on the functions of the corresponding protein products. Pathway analysis revealed that differentially expressed genes are enriched in pathways related to inflammation and immune responses. Quantitative real-time PCR analysis confirmed that the expressions of CXCL10, CXCL14, IL-18, IL-4RA, ABCA1, CCL4, IL-7R, CXCR4, TLR7, and CCL3 mRNAs were regulated by extracellular acidification.

CONCLUSIONS

The results of this study provide insights into the effects of acidic extracellular environments on macrophage gene expression.

OBJECTIVE

To explore the molecular mechanism of hepatitis B virus-related and hepatitis C virus-related hepatocellular carcinoma, samples from hepatitis B virus and hepatitis C virus infected patients and the normal were compared, respectively.

METHODS

In both experiments, genes with high value were selected based on a genome-wide relative significance and genome-wide global significance model. Co-expression network of the selected genes was constructed, and transcription factors in the network were identified. Molecular complex detection algorithm was used to obtain sub-networks.

RESULTS

Based on the new model, the top 300 genes were selected. Co-expression network was constructed and transcription factors were identified. We obtained two common genes FCN2 and CXCL14, and two common transcription factors RFX5 and EZH2. In hepatitis B virus experiment, cluster 1 and 3 had the higher value. In cluster 1, ten of the 17 genes and one transcription factor were all reported associated with hepatocellular carcinoma. In cluster 3, transcription factor ESR1 was reported related with hepatocellular carcinoma. In hepatitis C virus experiment, the value of cluster 3 and 4 was higher. In cluster 3, nine genes were reported to play a key role in hepatocellular carcinoma. In cluster 4, there were 5 genes in the 34 genes. To compare the relevance of a node in holding together communicating nodes, centralities based analysis was performed and we obtained some genes with high stress value.

CONCLUSIONS

The analysis above helped us to understand the pathogenesis of hepatitis B virus and hepatitis C virus associated hepatocellular carcinoma.

CXCL14 is a CXC-type chemokine acting on tissue macrophages, immature dendritic cells, natural killer cells, and epithelial tumor cells. It also serves as a metabolic regulator in obese mice by blunting insulin activity. In contrast to other CXC chemokines, it remains to be clarified how CXCL14 activates its putative receptors on the cell surface and whether it induces chemokinesis. This is mainly due to the insufficient sensitivity of currently available bioassays for CXCL14. In this study, we found that the anti-CXCL14 monoclonal antibody, MAB730, remarkably enhances the activities of CXCL14 in human monocytic leukemia-derived THP-1 cells and immature dendritic cells. MAB730 augmented CXCL14-mediated chemotaxis and chemokinesis with distinct dose requirement. Chemotaxis inducing activity was retained in the MAB730 F(ab')(2) fraction, but not in the Fab fraction, implying that ligand dimerization is involved in the MAB730-assisted enhancement of CXCL14 activity. In addition, MAB730 was more efficient than heparin at inhibiting CXCL14 binding to low affinity receptors on THP-1 cells. Finally, in vivo administration of MAB730 antibody into high fat diet-induced obese mice increased whole body insulin resistance and glucose intolerance. These unique properties of MAB730 will be useful for elucidating the molecular mechanism of cellular responses elicited by CXCL14.

To understand the molecular alterations driving the progression of ductal carcinoma in situ (DCIS), we compared patients with pure DCIS and patients with DCIS and synchronous invasive breast cancer (IBC). Twelve patients with extensive pure DCIS were included as a representation of indolent lesions with limited invasive capacity. These cases were matched with 12 patients with a limited DCIS component and IBC, representing lesions with a high invasive potential. Matching included age and surrogate DCIS subtypes. Gene expression profiling was performed on DCIS cells to identify transcriptional differences between these two groups. The identified genes were validated by immunohistochemistry. Nine genes showed significantly different expression. Most of these genes were highly expressed in DCIS samples with IBC, including PLAU (P = 0.002), COL1A1 (P = 0.006), KRT81 (P = 0.009), S100A7 (P = 0.015), SCGB1D2 (P = 0.023), KRT18 (P = 0.029), and NOTCH3 (P = 0.044), whereas EGFR and CXCL14 showed a higher expression in cases with pure DCIS (P = 0.015 and P = 0.028, respectively). This difference was only significant for SCGB1D2 (P = 0.009). Hierarchical clustering revealed distinct clustering of patients with and without invasion. Patients with pure DCIS have a different gene expression pattern as compared to patients with DCIS and synchronous IBC. These genes may pinpoint to driver pathway(s) that play an important role in DCIS progression.

CXCL14 is a CXC-type chemokine that exhibits chemotactic activity for immature dendritic cells, activated macrophages, and activated natural killer cells. However, its specific receptor and signaling pathway remain obscure. Recently, it was reported that CXCL14 binds to CXCR4 with high affinity and inhibits CXCL12-mediated chemotaxis. Furthermore, the CXCL14 C-terminal α-helical region is important for binding to its receptor. In this context, we chemically synthesized CXCL14 and its derivative with a one-pot method using N-sulfanylethylanilide peptide as a thioester equivalent. The synthetic CXCL14 proteins possessed inhibitory activities to CXCL12-mediated chemotaxis comparable with that of recombinant CXCL14. Moreover, we proved that chemically biotinylated CXCL14 binds to CXCR4 on cells by flow cytometry analysis.