Model establishment of prognostic-related immune genes in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck in the world. At present, the treatment methods include surgery, radiotherapy, and chemotherapy, but the 5-year survival rate is still not ideal and the quality of life of the patients is low. Due to the relative lack of immunotherapy methods, this study aims to build a risk prediction model of related immune genes, which can be used to effectively predict the prognosis of laryngeal cancer patients, and provide targets for subsequent immunotherapy. METHODS: We collected the 111 cases of laryngeal squamous cell carcinoma and 12 matched normal samples in the The Cancer Genome Atlas Database (TCGA) gene expression quantification database. The differentially expressed related immune genes were screened by R software version 3.5.2. The COX regression model of immune related genes was constructed, and the sensitivity and specificity of the model were evaluated. The risk value was calculated according to the model, and the risk curve was drawn to verify the correlation between related immune genes, risk score, and clinical traits. RESULTS: We selected 8 immune-related genes that can predict the prognosis of LSCC in a COX regression model and plotted the Kaplan-Meier survival curve. The 5-year survival rate of the high-risk group was 16.5% (95% CI: 0.059-0.459), and that of the low-risk group was 72.9% (95% CI: 0.555-0.956). The area under the receiver operating characteristic (ROC) curve was used to confirm the accuracy of the model (AUG = 0.887). After univariate and multivariate regression analysis, the risk score can be used as an independent risk factor for predicting prognosis. The risk score (P = .021) was positively correlated with the clinical Stage classification. CONCLUSION: We screened out 8 immune genes related to prognosis: RBP1, TLR2, AQP9, BTC, EPO, STC2, ZAP70, and PLCG1 to construct risk value models, which can be used to speculate the prognosis of the disease and provide new targets for future immunotherapy.

Cellular retinol-binding protein-1 expression increases with increasing clinical severity of alopecia areata.

BACKGROUND: Alopecia areata (AA) is multifactorial disease mostly autoimmune affecting anagen hair follicles. Many researchers hypothesize that adequate retinoic acid (RA) levels are important for proper hair follicle behavior. Previous animal studies revealed increase in RA synthesis proteins and decrease in RA degradation proteins in AA patients when compared with controls. OBJECTIVE: To evaluate cellular retinol-binding protein-1 expression in lesional skin of alopecia areata in comparison with controls, in an attempt to know its role in the pathogenesis of alopecia areata . METHODS: Immunohistochemical expression of cellular retinol-binding protein-1 CRBP1 was evaluated in skin biopsies taken from lesions of alopecia areata in 30 patients and 10 normal biopsy specimens taken from skin of healthy controls (HC) who were within the same age and sex. RESULTS: CRBP1 expression was significantly increased in lesional alopecia areata skin in comparison with normal skin of controls (P < 0.001*). Significant positive correlation was found between expression of CRBP-1 and percentage of hair loss in the scalp (SALT score; r = 0.840, P = <0.001). CONCLUSION: These results may enhance the idea of the possible role of CRBP1 in the pathogenesis of AA, and ensuring the importance of its level in AA treatment.

CRBP-1 over-expression is associated with poor prognosis in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is one of the most common malignancies of oral squamous cell carcinomas. Cellular retinol binding protein-1 (CRBP-1) as a carrier protein transports retinol from the liver storage site to peripheral tissue. Up-regulated expression of CRBP-1 is associated with some tumor types such as prostate cancer, breast cancer and ovarian cancer as reported, but its role in TSCC remains uncertain. METHODS: In this study, an integrated bioinformatics analysis based on the multiple cancer microarray data sets available from Oncomine database was conducted to view the differential expression of CRBP-1 between TSCC and the adjacent non-tumorous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB) and immunohistochemical (IHC) assays were performed to investigate CRBP-1 expression in 101 paraffin-embeded TSCC tissues and 48 pairs of freshly frozen tissues. Kaplan-Meier curve and univariate and multivariate Cox-regression analysis were used to estimate the association between CRBP-1 expression and patients' prognosis. Then western blotting, MTT, transwell migration and invasion assays were performed in TSCC cell lines to investigate the effects of CRBP-1 on cellular proliferation and invasion. RESULTS: Compared with the matched adjacent non-tumorous tissues, the expression of CRBP-1 was significantly up-regulated in TSCC tissues, which correlated with the differentiation state (P = 0.003), N classification (P = 0.048), the clinical stage (P = 0.048) and death (P = 0.001). The Kaplan-Meier curve showed that TSCC patients with higher CRBP-1 expression levels had lower overall survival rates than those with lower CRBP-1 expression levels. A univariate and multivariate analysis demonstrated that CRBP-1 was an independent prognostic factor (P < 0.05). Furthermore, we knocked down CRBP-1 expression and observed that TSCC cell proliferation and invasion in vitro were significantly blocked, as determined by MTT and transwell assays. CONCLUSIONS: Up-regulated expression of CRBP-1 is associated with poor prognosis in TSCC, so it might potentially serve as an additional prognostic marker, and the inhibition of CRBP-1 might provide new therapeutic approaches for TSCC.

Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry.

Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range (~1-10 µM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system. Graphical Abstract ᅟ.

RBP2 induces stem-like cancer cells by promoting EMT and is a prognostic marker for renal cell carcinoma.

Renal cell carcinoma (RCC), one of the most common kidney cancers, has a poor prognosis. Epithelial to mesenchymal transition (EMT) is a hallmark of carcinoma invasion and metastasis. Several studies have examined the molecular regulation of EMT, but the relationship between histone demethylases and EMT is little understood. In this study, we investigated the role of retinoblastoma-binding protein-2 (RBP2), a histone demethylase that is highly expressed in RCC and is positively correlated with poor RCC prognosis in the regulation of EMT. We found that ectopic overexpression of RBP2 can induce cancer stem cell-like (CSC) phenotypes through EMT in RCC cells by converting them to a more mesenchymal phenotype. This results in increased resistance to apoptosis, which leads to enhanced tumor growth in xenograft models. Together, our data show that RBP2 is an epigenetic regulator that has an important role in the initiation of CSC phenotypes through EMT, leading to tumor progression. RBP2 is also a novel biomolecule for RCC diagnosis, and prognosis and may be a therapeutic target.

Premature differentiation of vascular smooth muscle cells in human congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterized by the herniation of abdominal organs into the chest cavity. The high mortality and morbidity of CDH patients are primarily caused by the associated pulmonary hypertension (PH), characterized by the thickening of the vascular media and adventitia. The media consist of heterogeneous populations of vascular smooth muscle cells (VSMC), ranging from synthetic to the characteristic contractile cells. VSMCs are influenced by developmental and environmental cues and may play a role in the development of the structural changes observed in CDH patients. Therefore, we hypothesized that the distribution of the VSMC populations may already be different at the origin of CDH development. METHODOLOGY: We analyzed the protein expression of specific markers associated with synthetic and contractile VSMC phenotypes in human lungs at different developmental stages. Next, we compared lungs of premature and term CDH patients, as well as patients with lung hypoplasia due to renal agenesis or PROM, with age-matched controls. RESULTS: Synthetic and contractile VSMCs are distributed in a temporal and spatial specific pattern along the proximodistal axis of the lung. CDH patients have more abundant contractile VSMCs which are also more distally distributed. This different distribution pattern is already observed from 19 weeks of gestation onwards. CONCLUSION: Our data suggest that the more extensive distribution of contractile VSMCs is associated with an early maturation of the pulmonary vasculature, contrasting the concept that CDH might be the result of delayed maturation of the epithelium.

Identification of retinol binding protein 1 promoter hypermethylation in isocitrate dehydrogenase 1 and 2 mutant gliomas.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 (IDH1) and associated CpG island hypermethylation represent early events in the development of low-grade gliomas and secondary glioblastomas. To identify candidate tumor suppressor genes whose promoter methylation may contribute to gliomagenesis, we compared methylation profiles of IDH1 mutant (MUT) and IDH1 wild-type (WT) tumors using massively parallel reduced representation bisulfite sequencing. METHODS: Reduced representation bisulfite sequencing was performed on ten pathologically matched WT and MUT glioma samples and compared with data from a methylation-sensitive restriction enzyme technique and data from The Cancer Genome Atlas (TCGA). Methylation in the gene retinol-binding protein 1 (RBP1) was identified in IDH1 mutant tumors and further analyzed with primer-based bisulfite sequencing. Correlation between IDH1/IDH2 mutation status and RBP1 methylation was evaluated with Spearman correlation. Survival data were collected retrospectively and analyzed with Kaplan-Meier and Cox proportional hazards analysis. All statistical tests were two-sided. RESULTS: Methylome analysis identified coordinated CpG island hypermethylation in IDH1 MUT gliomas, consistent with previous reports. RBP1, important in retinoic acid metabolism, was found to be hypermethylated in 76 of 79 IDH1 MUT, 3 of 3 IDH2 MUT, and 0 of 116 IDH1/IDH2 WT tumors. IDH1/IDH2 mutation was highly correlated with RBP1 hypermethylation (n = 198; Spearman R = 0.94, 95% confidence interval = 0.92 to 0.95, P < .001). The Cancer Genome Atlas showed IDH1 MUT tumors (n = 23) to be RBP1-hypermethylated with decreased RBP1 expression compared with WT tumors (n = 124). Among patients with primary glioblastoma, patients with RBP1-unmethylated tumors (n = 102) had decreased median overall survival compared with patients with RBP1-methylated tumors (n = 22) (20.3 months vs 36.8 months, respectively; hazard ratio of death = 2.48, 95% confidence interval = 1.30 to 4.75, P = .006). CONCLUSION: RBP1 promoter hypermethylation is found in nearly all IDH1 and IDH2 mutant gliomas and is associated with improved patient survival. Because RBP1 is involved in retinoic acid synthesis, our results suggest that dysregulation of retinoic acid metabolism may contribute to glioma formation along the IDH1/IDH2-mutant pathway.

Correlation between expression of cellular retinol-binding protein 1 and its methylation status in larynx cancer.

AIMS: The authors have previously reported that cellular retinol-binding protein 1 (CRBP1) gene gain and its expression correlated significantly with survival in laryngeal carcinoma patients. The authors hypothesised that inactivation of the CRBP1 gene through CpG methylation is associated with patient status and gene expression. In this work, the authors determine the expression and methylation status of the CRBP1 gene and its correlation with clinical variables of laryngeal carcinoma patients. METHODS: The CRBP1 gene methylation promoter was assessed by methylation specific PCR analysis in tissue samples from larynx cancer specimens and its expression was assessed by immunohistochemistry on paraffin embedded tissue using tissue microarray. The results were then compared with the clinical pathological variables and outcome measures. The study included 46 samples from patients with non-metastatic squamous cell carcinoma of the larynx without any previous oncological treatments. RESULTS: Lack of CRBP1 expression was seen in 17 of the 46 laryngeal carcinoma samples, while the remaining 29 samples showed increased expression. Significant associations were found between CRBP1 expression and methylation and patient status. There was a tendency for association in all clinical stages of the disease. CRBP1 gene expression and its unmethylated promoter correlated significantly with survival (p<0.05). CONCLUSIONS: An early event of larynx cancer could be CRBP1 expression related to unmethylation of the promoter region. These features could also be associated with good response and survival. The authors hypothesised that increased expression and unmethylated promoter of the CRBP1 gene could be considered as markers for larynx cancer.

Microarray analyses of oral punch biopsies from acute myeloid leukemia (AML) patients treated with chemotherapy.

OBJECTIVE: Understanding the pathogenesis of chemotherapy-induced oral mucositis (CIOM) is vital to develop therapies for this common, dose-limiting side effect of cancer treatment. We investigated molecular events in CIOM from buccal mucosa tissue collected before and 2 days after chemotherapy from patients with acute myeloid leukemia (AML) and healthy controls by microarray analysis. METHODS: Microarray analysis was performed using Human Genome U133 Plus 2.0 Array on buccal mucosa punch biopsies from patients with AML before (n = 4) or after chemotherapy (n = 4), and from healthy controls (n = 3). Following Robust Multichip Average (RMA) normalization, we applied Linear Models for Microarray data (LIMMA) and Significance Analysis of Microarrays (SAM) for data analysis using the TM4/TMeV v4.5.1 program. RESULTS: LIMMA and SAM identified genes potentially affected by the presence of AML, including homeodomain-interacting protein kinase 1 (HIPK1), mex-3 homolog D (MEX3D), and genes potentially affected by chemotherapy, including argininosuccinate synthase 1 (ASS1), notch homolog 1 (NOTCH1), zinc transporter ZIP6 (SLC39A6), and TP53-regulated inhibitor of apoptosis 1 (TRIAP1). The expression of 2 genes with potential biological significance in oral mucositis, ASS1 and SLC39A6 (alias LIV-1), was confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). CONCLUSIONS: Our results suggest that AML-specific deregulated immune responses and inflammatory tissue damage to the oral mucosa caused by chemotherapy may not be overcome by the natural cellular repair processes and therefore contribute to CIOM.

Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver.

Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.

Expression analysis and RNA localization of PAI-RBP1 (SERBP1) in epithelial ovarian cancer: association with tumor progression.

OBJECTIVE: The plasminogen activator system (PA) plays an important role in invasion and metastasis of solid tumors. The PA Inhibitor type 1 (PAI-1) is the main physiologic regulator of plasminogen activation. A recently characterized protein, PAI-RBP1 (PAI-1 mRNA Binding Protein 1), appears to regulate the stability of PAI-1 mRNA. Expression of PAI-RBP1 (the new, approved gene symbol is SERBP1) has not been previously analyzed in human tumors. We present herein for the first time expression analysis of PAI-RBP1 in epithelial ovarian cancer. METHODS: PAI-RBP1 was identified as gene overexpressed in ovarian cancer by an in silico approach using EST database mining. A thorough expression analysis of PAI-RBP1 and PAI-1 was performed in normal ovary (n=4), benign (n=6) and malignant (n=42) ovarian lesions using non-radioactive RNA in situ hybridization and immunohistochemistry, respectively. RESULTS: PAI-RBP1 mRNA and PAI-1 were significantly overexpressed in tumor epithelial cells as compared to benign and normal ovarian tissue. A significant correlation between PAI-RBP1 expression and advanced disease stage (FIGO) was found (p=0.042). CONCLUSION: In ovarian cancer, PAI-RBP1 is significantly overexpressed in tumor epithelial cells, suggesting a biological role in tumor invasion and metastasis. Its expression is higher in advanced disease, thus the prognostic significance of PAI-RBP1 in ovarian cancer remains to be evaluated in further studies.