Spleen Associated Tyrosine Kinase Research Articles

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of β-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with β-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to β-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1β, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides β-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. Oral epithelial cells express Dectin-1 and recognize β-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

T-independent B-cell effect of agents associated with swine grower-finisher diarrhea.

Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.

A novel in silico scaffold-hopping method for drug repositioning in rare and intractable diseases

In the field of rare and intractable diseases, new drug development is difficult and drug repositioning (DR) is a key method to improve this situation. In this study, we present a new method for finding DR candidates utilizing virtual screening, which integrates amino acid interaction mapping into scaffold-hopping (AI-AAM). At first, we used a spleen associated tyrosine kinase inhibitor as a reference to evaluate the technique, and succeeded in scaffold-hopping maintaining the pharmacological activity. Then we applied this method to five drugs and obtained 144 compounds with diverse structures. Among these, 31 compounds were known to target the same proteins as their reference compounds and 113 compounds were known to target different proteins. We found that AI-AAM dominantly selected functionally similar compounds; thus, these selected compounds may represent improved alternatives to their reference compounds. Moreover, the latter compounds were presumed to bind to the targets of their references as well. This new “compound-target” information provided DR candidates that could be utilized for future drug development.

Analysis of Genes Related to Invadopodia Formation and CTTN in Oral Squamous Cell Carcinoma-A Systematic Gene Expression Analysis.

Successful treatment for any type of carcinoma largely depends on understanding the patterns of invasion and migration. For oral squamous cell carcinoma (OSCC), these processes are not entirely understood as of now. Invadopodia and podosomes, called invadosomes, play an important role in cancer cell invasion and migration. Previous research has established that cortactin (CTTN) is a major inducer of invadosome formation. However, less is known about the expression patterns of CTTN and other genes related to it or invadopodia formation in OSCC during tumor progression in particular. In this study, gene expression patterns of CTTN and various genes (n = 36) associated with invadopodia formation were analyzed to reveal relevant expression patterns and give a comprehensive overview of them. The genes were analyzed from a whole genome dataset of 83 OSCC samples relating to tumor size, grading, lymph node status, and UICC (Union for Internatioanl Cancer Control). The data revealed significant overexpression of 18 genes, most notably CTTN, SRC (SRC proto-onocogene, non-receptor tyrosine kinase), EGFR (epidermal growth factor receptor), SYK (spleen associated tyrosine kinase), WASL (WASP like actin nucleation promotion factor), and ARPC2 (arrestin beta 1) due to their significant correlation with further tumor parameters. This study is one of the first to summarize the expression patterns of CTTN and related genes in a complex group of OSCC samples.

Conserved transcriptional programming across sex and species after peripheral nerve injury predicts treatments for neuropathic pain.

Chronic pain is a devastating problem affecting one in five individuals around the globe, with neuropathic pain the most debilitating and poorly treated type of chronic pain. Advances in transcriptomics have contributed to cataloguing diverse cellular pathways and transcriptomic alterations in response to peripheral nerve injury but have focused on phenomenology and classifying transcriptomic responses. To identifying new types of pain-relieving agents, we compared transcriptional reprogramming changes in the dorsal spinal cord after peripheral nerve injury cross-sex and cross-species, and imputed commonalities, as well as differences in cellular pathways and gene regulation. We identified 93 transcripts in the dorsal horn that were increased by peripheral nerve injury in male and female mice and rats. Following gene ontology and transcription factor analyses, we constructed a pain interactome for the proteins encoded by the differentially expressed genes, discovering new, conserved signalling nodes. We investigated the interactome with the Drug-Gene database to predict FDA-approved medications that may modulate key nodes within the network. The top hit from the analysis was fostamatinib, the molecular target of which is the non-receptor spleen associated tyrosine kinase (Syk), which our analysis had identified as a key node in the interactome. We found that intrathecally administrating the active metabolite of fostamatinib, R406 and another Syk inhibitor P505-15, significantly reversed pain hypersensitivity in both sexes. Thus, we have identified and shown the efficacy of an agent that could not have been previously predicted to have analgesic properties.

Interpretable deep learning translation of GWAS and multi-omics findings to identify pathobiology and drug repurposing in Alzheimer's disease.

Translating human genetic findings (genome-wide association studies [GWAS]) to pathobiology and therapeutic discovery remains a major challenge for Alzheimer's disease (AD). We present a network topology-based deep learning framework to identify disease-associated genes (NETTAG). We leverage non-coding GWAS loci effects on quantitative trait loci, enhancers and CpG islands, promoter regions, open chromatin, and promoter flanking regions under the protein-protein interactome. Via NETTAG, we identified 156 AD-risk genes enriched in druggable targets. Combining network-based prediction and retrospective case-control observations with 10 million individuals, we identified that usage of four drugs (ibuprofen, gemfibrozil, cholecalciferol, and ceftriaxone) is associated with reduced likelihood of AD incidence. Gemfibrozil (an approved lipid regulator) is significantly associated with 43% reduced risk of AD compared with simvastatin using an active-comparator design (95% confidence interval 0.51-0.63, p<0.0001). In summary, NETTAG offers a deep learning methodology that utilizes GWAS and multi-genomic findings to identify pathobiology and drug repurposing in AD.

Onc201 reduces osteoclastogenesis and prevents ovariectomy-induced bone loss via inhibiting RANKL-induced NFATc1 activation and the integrin signaling pathway

Osteoporosis is an osteolytic disease with a disrupted balance between the resorption and formation of bone as well as bone microstructure degeneration, leading to bone loss and increased fracture risk, which greatly affects patients’ quality of life. Currently, inhibition of osteoclast bone resorption remains the mainstream treatment for osteoporosis. Onc201, a new compound, induces the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and has an efficient anticancer effect in clinical trials. However, its effects on osteolytic disease and the mechanism of action are unclear. We examined the effect of Onc201 on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts via Cell Counting Kit-8, bone resorption assay, luciferase reporter assay, immunofluorescence staining, calcium ion intensity assay and employed an ovariectomy model to investigate the effect of Onc201 on osteoporosis in the mice. Results showed that Onc201 inhibited the function and formation of osteoclasts induced by RANKL in a manner that was dependent on time and concentration, and did not cause cytotoxicity. Mechanistically, Onc201 inhibited osteoclast-relevant genes and NFATc1 expression, the main transcriptional regulatory factor of the formation of osteoclasts induced by RANKL; meanwhile, downregulating the expressions of the osteoclast cytoskeleton key signal molecules integrin αvβ3, focal adhesion kinase (FAK), c-Src, and spleen-associated tyrosine kinase (SYK). In addition, Onc201 had a protective effect on the mouse model of bone loss caused by ovariectomy-induced estrogen deficiency, which is consistent with the in vitro results. Our findings suggest that the new small-molecular compound Onc201 has the potential to prevent osteoclast-related osteolytic diseases.

American Joint Committee on Cancer Tumor Staging System Predicts the Outcome and Metastasis Pattern in Conjunctival Melanoma

To assess the predictive value of the tumor staging system in the AJCC Cancer Staging Manual, Eighth Edition, and histologic features for outcomes and metastasis patterns in conjunctival melanoma (CM). Retrospective, single-center cohort study. Eighty-three patients with CM were treated at Shanghai Ninth People's Hospital between 2000 and2021. We reviewed the clinical and histologic parameters and used Kaplan-Meier survival curves and Cox proportional hazards regression models for risk factor analyses. Time to nodal/distant metastasis, disease-specific survival, metastatic pattern, and metastatic site. At presentation, 5 patients (6%) had clinical tumor (cT)1 disease, 34 patients (41%) had cT2 disease, and 44 patients (53%) had cT3 disease. Four patients (5%) had nodal metastasis (N1), and none had distant metastasis (M1). During follow-up, 12 patients (14%) developed nodal metastasis, 29 patients (35%) developed distant metastasis, and 26 patients (31%) died of disease. The brain, liver, and lung were common distant metastasis sites. Higher cT category was associated with increased risks of distant metastasis (P < 0.001) and disease-specific death (P= 0.002). The separate analysis of primary and recurrent tumors at presentation showed that the patients with cT3 tumors had a higher risk of distant metastasis than those with cT2 tumors. Greater tumor thickness, ulceration, and the presence of regression were correlated with distant metastasis. Previously unreported mutations were detected in the tumor suppressor genes FAT atypical cadherin 4 (FAT4) and spleen associated tyrosine kinase (SYK). Among the 29 patients who developed distant metastasis, we analyzed 2 patterns of metastasis: Eleven patients (38%) developed nodal metastasis before distant metastasis, and 18 patients (62%) developed distant metastasis without previously known nodal metastasis. The patients with cT3 tumors were more likely to follow the latter metastasis pattern (P= 0.02). Conjunctival melanoma presented with mostly advanced stages and high rates of distant metastasis in the current Chinese cohort. This study confirmed the prognostic value of the tumor staging system in the AJCC Cancer Staging Manual, Eighth Edition, for Chinese patients. Histologic features, such as tumor thickness and ulceration, should be emphasized when assessing prognosis and guiding the treatment of CM.

DOCK2 regulates antifungal immunity by regulating RAC GTPase activity.

Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.

Molecular Structure, Spectroscopic Investigation, Docking and in Vitro Cytotoxicity Studies of 7-Methoxycoumarin as anti-Leukemia Agent

The vibrational assignments of 7-methoxycoumarin (7MC), a potential anticancer agent, have been studied by the experimental (FTIR and FT-Raman) and theoretical methods. The density functional theory DFT- B3LYP technique with cc-pVTZ basis set is utilized to examine the molecular geometry, electrostatic potential (MEP), infrared intensities, Raman activities, Mulliken charge and thermodynamic limits. The molecular charge exchange in orbitals has been examined by natural bond orbital (NBO) analysis. HOMO-LUMO transition and the UV absorption by using TD-DFT approach have been investigated. The molecular docking studies have been executed for interaction of 7MC with the sites of human Bruton tyrosine kinase (BTK), Phosphoinositide 3'-kinase (PI3K) and Spleen Associated Tyrosine Kinase (SYK). MTT assay is used to study the anticancer activity of 7MC through five human cancer cell lines (A-549, HEPG2, HEp2, MCF-7, and HL-60). The results suggested that the 7MC can have possibly been used as an agent of anti-leukemia efficacy.

Transcriptome sequencing identifies genes associated with invasion of ovarian cancer.

ObjectiveTo identify key genes in ovarian cancer using transcriptome sequencing in two cell lines: MCV152 (benign ovarian epithelial tumour) and SKOV-3 (ovarian serous carcinoma).MethodsDifferentially expressed genes (DEGs) between SKOV-3 and MCV152 were identified. Candidate genes were assessed for enrichment in gene ontology function and Kyoto Encyclopaedia of Genes and Genomes pathway. Candidate gene expression in SKOV-3 and MCV152 cells was validated using Western blots.ResultsA total of 2020 upregulated and 1673 downregulated DEGs between SKOV3 and MCV152 cells were identified that were significantly enriched in the cell adhesion function. Upregulated DEGs, such as angiopoietin 2 (ANGPT2), CD19 molecule (CD19), collagen type IV alpha 3 chain (COL4A3), fibroblast growth factor 18 (FGF18), integrin subunit beta 4 (ITGB4), integrin subunit beta 8 (ITGB8), laminin subunit alpha 3 (LAMA3), laminin subunit gamma 2 (LAMC2), protein phosphatase 2 regulatory subunit Bgamma (PPP2R2C) and spleen associated tyrosine kinase (SYK) were significantly involved in the extracellular matrix-receptor interaction pathway. Downregulated DEGs, such as AKT serine/threonine kinase 3 (AKT3), collagen type VI alpha 1 chain (COL6A1), colony stimulating factor 3 (CSF3), fibroblast growth factor 1 (FGF1), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 11 (ITGA11), MYB proto-oncogene, transcription factor (MYB), phosphoenolpyruvate carboxykinase 2, mitochondrial (PCK2), placental growth factor (PGF), phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), serum/glucocorticoid regulated kinase 1 (SGK1), toll like receptor 4 (TLR4) and tumour protein p53 (TP53) were involved in PI3K-Akt signalling. Expression of these DEGs was confirmed by Western blot analyses.ConclusionCandidate genes enriched in cell adhesion, extracellular matrix–receptor interaction and PI3K-Akt signalling pathways were identified that may be closely associated with ovarian cancer invasion and potential targets for ovarian cancer treatment.

Novel blood-based biomarker predicting severe toxicity in melanoma anti-CTLA-4 immunotherapy treatment.

3077 Background: Immune checkpoint inhibition (ICI) has improved clinical outcomes of metastatic melanoma (MM). However, 65-80% of treated patients experience immune-related adverse events (irAEs), urging for the availability of personalized and easy-access clinical biomarkers. We have previously shown that germline genetics related to host immunity affects ICI response and MM survival. In this study, we investigated if germline immune-related expression quantitative trait loci (ieQTLs) may predict ICI-induced irAEs in MM. Methods: Through a comprehensive interrogation of a healthy twin-cohort expression dataset (MuTHER), we identified 40 ieQTLs most significantly associated with the expression of 382 immune-related genes. These germline variants were genotyped using the MassARRAY system in anti-CTLA-4-treated MM patients, collected as part of a multi-institutional collaboration. Using multivariable logistic regression models, we tested the association of 40 ieQTLs with irAEs in a discovery cohort of 97 MM patients followed by a validation in additional cohort of 97 anti-CTLA-4 treated patients. Results: We found rs7036417 significantly associated with severe anti-CTLA-4 irAEs in the discovery (OR = 6.18; 95%CI = 1.61-23.74; p = 0.007) and validation (OR = 6.73, 95%CI = 1.42-31.86; p = 0.02) cohorts. Pooled analysis showed that carriers of two rs7036417 alternate alleles (TT) have a 6-fold increased risk of developing severe irAEs (OR = 6.11; 95% = 2.26-16.56;p = 0.0003). This association was not observed with ICI response or survival. The alternate allele of rs7036417 is associated with higher expression of SYK (spleen-associated tyrosine kinase), suggesting that elevated SYK contributes to developing severe irAEs. Conclusions: We report that rs7036417, an ieQTL in SYK, associates with an increased risk of severe irAEs, independent of ICI efficacy. SYK plays an important role in B-cell/T-cell expansion and increased pSYK has been reported in patients with rheumatoid arthritis or systemic lupus erythematosus. Based on our data, the over-expression of SYK likely explains the biological mechanisms of the association between rs7036417 and anti-CTLA4 irAEs. These findings propose a novel blood-based baseline biomarker stratifying the patients at increased risk of severe irAEs, with a clinical effect substantially surpassing those observed for currently available predictors. Our ongoing studies are currently investigating SYK eQTL as a novel target in toxicity-reducing therapies.

Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+] i ), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+] i Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Methylation of selected CpG islands involved in the transcription of myeloperoxidase and superoxide dismutase 2 in neutrophils of periparturient and mid-lactation cows

It is generally accepted that intracellular killing of microorganisms by production of reactive oxygen species (ROS) in the phagosome of the neutrophil is an important arm of innate defense. High-producing dairy cows are prone to periparturient metabolic and infectious diseases. Both myeloperoxidase (MPO) activity and ROS production decrease the day of parturition. Several studies have demonstrated changes in the expression of genes involved in, for example, metabolism and defense in the circulating neutrophil during peripartum. In this study, we wanted to further characterize the periparturient neutrophil in terms of its oxidative killing capacity by analyzing the oxidative burst at 3 levels. First, the ROS phenotype was evaluated using chemiluminescence. The cows (sampled within 24 h after parturition and at 135 d in milk) showed a significantly slower production of ROS at parturition. Both primiparous (n = 13) and multiparous (n = 12) cows were included in this study, but parity did not affect the kinetics of ROS production. Second, the expression of 11 genes involved in ROS production was measured in the same cows: cytochrome b-245 α and β chain (CYBA, CYBB; coding for membrane-bound constituents of NADPH oxidase); neutrophil cytosolic factors 1, 2, and 4 (NCF1, NCF2, and NCF4); Rac family small GTPase 1 and 2 (RAC1 and RAC2; coding for regulatory proteins of NADPH oxidase); superoxide dismutase 2 (SOD2); catalase (CAT); myeloperoxidase (MPO; coding for enzymes involved in metabolizing downstream ROS); and spleen-associated tyrosine kinase (SYK; involved in signaling). During peripartum, a shift in expression in the oxidative killing pathway was observed, characterized by a downregulation of MPO and a simultaneous upregulation of the genes coding for NADPH oxidase. Third, as total DNA methylation is known to change during pregnancy, we investigated whether the observed differences were due to different methylation patterns. Promotor regions initiate transcription of particular genes; therefore, we analyzed the methylation status in annotated CpG islands of MPO and SOD2, 2 genes with a significant difference in expression between both lactation stages. The differences in methylation of these CpG islands were nonsignificant. High-throughput techniques may be necessary to obtain more detailed information on the total DNA methylation dynamics in bovine neutrophils and increase our understanding of how gene expression is controlled in neutrophils.

Association between methylation of DNA damage response-related genes and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride

In the present study, we investigated the association between methylation of DNA damage response-related genes such as cyclin-dependent kinase inhibitor (CDKN)2A, Ras association (RalGDS/AF-6) domain family member (RASSF)1A, O6-methylguanine DNA methyltransferase (MGMT), Kirsten rat sarcoma viral oncogene homolog (KRAS), and spleen-associated tyrosine kinase (SYK) and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride (VC). Sixty-four healthy rats were randomly divided into three VC exposure groups (5, 25, and 125 mg/kg) and an untreated negative control group (n = 16 each). VC was administered by intraperitoneal injection every other day for a total of three times a week. Eight randomly selected rats from each group were sacrificed at the end of 6 and 12 weeks, and liver tissue was harvested for the comet assay and for assessment of DNA methylation level and mRNA expression of related genes by PCR. Overall methylation levels in the genome of hepatocytes in VC-exposed rats were higher than those in the control group at 6 and 12 weeks (P < 0.05), although no differences were observed with regarding to dose (P > 0.05). After 12 weeks of exposure, differences in the methylation of RASSF1A and MGMT promoter regions were observed between the high-dose group and other groups (P < 0.05), whereas no differences were observed for the KRAS, SYK, and CDKN2A promoters (P > 0.05). These results suggest that DNA damage and increased genome-wide methylation are biomarkers for VC exposure and that RASSF1A and MGMT promoter methylation is related to the carcinogenic mechanism of VC.

Lymphatics-associated genes are downregulated at transcription level in non-small cell lung cancer.

The present study aimed to verify a possibility of ongoing lymphangiogenesis in non-small cell lung cancer (NSCLC) via examination of mRNA levels of a number of lymphangiogenesis-associated genes in tumors. It was hypothesized that transcriptional activation of these genes would occur in tumors that stimulate new lymphatic vessel formation. The study was performed on 140 pairs of fresh-frozen surgical specimens of cancer and unaffected lung tissues derived from NSCLC stage I-IIIA patients. mRNA levels were evaluated with the reverse transcription-quantitative polymerase chain reaction method and expressed as fold change differences between the tumor and normal tissues. Possible associations between expression and patient clinicopathological characteristics and survival were analyzed. In the NSCLC tissue samples, vascular endothelial growth factor (VEGF) C, VEGFD, VEGFR3, VEGFR2, VEGFR1, lymphatic vessel endothelial hyaluronan receptor 1, integrin subunit α 9, FOX2, neuropilin 2, fibroblast growth factor 2 genes were significantly downregulated (P<0.001 for all) compared with matched normal lung tissues, whereas mRNA levels for VEGFA, spleen associated tyrosine kinase, podoplanin, and prospero homeobox 1 genes were similar in both tissues. Neither lymph node status, nor disease pathological stage influenced expression, whereas more profound suppression of gene activities appeared to occur in squamous cell carcinomas compared with adenocarcinomas. The VEGFR1 mRNA expression level was significantly connected with patient survival in the univariate analysis, and was an independent prognostic factor for overall survival in the multivariate Cox's proportional hazards model (HR 2.103; 95% confidence interval: 1.005-4.401; P=0.049). The results support a hypothesis of absence of new lymphatic vessel formation inside growing NSCLC tumor mass, however do not exclude a possibility of lymphangiogenesis in narrow marginal tumor parts.

Cigarette smoke-induced EGFR activation promotes epithelial mesenchymal migration of human retinal pigment epithelial cells through regulation of the FAK-mediated Syk/Src pathway.

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is inevitable change of age-related macular degeneration (AMD). Smoking is a major risk factor for the development of EMT in several diseases, including lung cancer. Cigarette smoke-induced stress promotes the production of epidermal growth factor (EGF) in RPE cells. However, the underlying signaling pathways induced by aberrant EGF receptor (EGFR) expression in cigarette smoke-exposed RPE cells remain largely unknown. In the present study, the morphological transformation and production of EMT-associated cytokines were investigated to analyze the effect of smoking on the retina. Furthermore, EGF-treated or cigarette smoke-exposed RPE cells, as well as the downstream targets of EGFR, were investigated to identify the key molecules involved in EMT of cigarette smoke-stimulated RPE cells via immunoblotting. Exposure of RPE cells to cigarette smoke extract (CSE) induced secretion of VEGF and TGF-β1, and increased the expression of EMT markers. CSE-mediated focal adhesion kinase (FAK) activation resulted in the phosphorylation and activation of spleen associated tyrosine kinase (Syk)/Src proto-oncogene, non-receptor tyrosine kinase (Src), leading to migration and invasion of RPE cells. Knockdown of FAK or pharmacological inhibition of Syk/Src abrogated CSE-mediated VEGF and TGF-β1 production and blocked the phosphorylation of Smad2/3 in CSE-stimulated RPE cells. Erlotinib (an EGFR inhibitor) suppressed EGF and CSE-mediated switch from an epithelial to mesenchymal phenotype. Baicalein, an inhibitor of 12/15-lipooxygenase, also efficiently suppressed CSE-induced EMT processes by inhibiting EGFR-associated downstream signaling transduction. The results identified a novel signaling pathway mediated by EGFR in CSE-activated RPE cells, and suggest baicalein as a potential new therapeutic drug for CSE-associated retinopathy.

Molecular mechanisms of breast cancer metastasis by gene expression profile analysis.

Metastasis is the main cause of breast cancer-related mortalities. The present study aimed to uncover the relevant molecular mechanisms of breast cancer metastasis and to explore potential biomarkers that may be used for prognosis. Expression profile microarray data GSE8977, which contained 22 stroma samples (15 were from normal breast and 7 were from invasive ductal carcinoma tumor samples), were obtained from the Gene Expression Omnibus database. Following data preprocessing, differentially expressed genes (DEGs) were selected based on analyses conducted using the linear models for microarray analysis package from R and Bioconductor software. The resulting data were used in subsequent function and pathway enrichment analyses, as well as protein-protein interaction (PPI) network and subnetwork analyses. Transcription factors (TFs) and tumor-associated genes were also identified among the DEGs. A total of 234 DEGs were identified, which were enriched in immune response, cell differentiation and cell adhesion-related functions and pathways. Downregulated DEGs included TFs, such as the proto-oncogene SPI1, pre-B-cell leukemia homeobox 3 (PBX3) and lymphoid enhancer-binding factor 1 (LEF1), as well as tumor suppressors (TSs), such as capping actin protein, gelsolin like (CAPG) and tumor protein p53-inducible nuclear protein 1 (TP53INP1). Upregulated DEGs also included TFs and tumor suppressors, consisting of transcription factor 7-like 2 (TCF7L2) and pleiomorphic adenoma gene-like 1 (PLAGL1). DEGs that were identified at the hub nodes in the PPI network and the subnetwork were epidermal growth factor receptor (EGFR) and spleen-associated tyrosine kinase (SYK), respectively. Several genes crucial in the metastasis of breast cancer were identified, which may serve as potential biomarkers, many of which were associated with cell adhesion, proliferation or immune response, and may influence breast cancer metastasis by regulating these function or pathways.

Intestinal Fungal Dysbiosis Is Associated With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats

Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble β-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (exvivo) and the human mast cell line HMC-1. α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble β-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate β-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble β-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.

Analysis of gene expression profiling variations induced by hsa‑miR‑145‑5p‑overexpression in laryngeal squamous cell carcinoma cell line Tu‑177.

The present study aimed to investigate the variations of the gene network and biological functions induced by hsa-miR-145-5p in the laryngeal squamous cell carcinoma (LSCC) cell line Tu-177. A hsa-miR-145-5p-overexpressed Tu-177 cell model was established, and the gene expression microarray data of miR-145-5p-overexpressed cells and negative control (NC) cells were analyzed. The differentially expressed genes (DEGs) between two groups were identified, and their potential functions were predicted by functional enrichment analysis. Furthermore, the targets of miR-145-5p were identified from the DEGs, and their potential functions and protein-protein interactions (PPIs) were analyzed. The mRNA expressions of acetyl-CoA carboxylase β (ACACB), fibroblast growth factor receptor 1 (FGFR1), protein phosphatase 3 catalytic subunit a (PPP3CA) and spleen associated tyrosine kinase (SYK), were analyzed via quantitative polymerase chain reaction. A total of 1,501 upregulated and 887 downregulated genes were identified in the hsa-miR-145-5p-overexpressed Tu-177 cells, compared with the NC cells. Of these DEGs, 164 upregulated and 221 downregulated genes were predicted to be targeted by hsa-miR-145-5p. The upregulated target genes were primarily associated with functions of immunity, whereas the downregulated target genes were significantly enriched in the p53 signaling pathway. In the PPI network consisting of 267 target genes, the upregulated ACACB had the greatest degree and interacted with downregulated genes including PPP3CA and SYK, in addition to upregulated genes, including FGFR1. The mRNA expressions of ACACB and FGFR1were markedly enhanced in miR-145-5p-overexpressed Tu-177 cells, whereas overexpressing miR-145-5p significantly reduced mRNA expression of PPP3CA and SYK. hsa-miR-145-5p may exhibit an anticancer role in LSCC via regulating multiple cell processes, including cell proliferation and invasion, fatty acid metabolism, immunity and p53 signaling pathway. These findings provide novel information for the future investigation of miR-145-5p functions in LSCC.