Urinary orosomucoid 1 protein to creatinine ratio as a potential biomarker for early screening of kidney impairment in type-2 diabetes patients.

BACKGROUND: Early screening of diabetic kidney disease (DKD) remains a major challenge. Our aim was to evaluate the value of urinary orosomucoid 1 protein (UORM1) in early renal impairment screening in type-2 diabetes patients. METHODS: The concentration of UORM1, the UORM1-to-creatinine ratio (UORM1CR), the urinary albumin-to-creatinine ratio (ACR), the alpha-1-microglobulin-to-creatinine ratio (A1MCR) and estimated glomerular filtration rate (eGFR) were measured in 406 type-2 diabetes patients. Any positive values for ACR, A1MCR and/or eGFR were considered as indicative of renal impairment. RESULTS: On average, the levels of UORM1 and UORM1CR were about seven times higher in subjects with renal injury than in those without. Both UORM1 and UORM1CR, when adjusted via logarithm-transformation, were significantly related to ACR, A1MCR and eGFR levels. The highest correlation was observed between UORM1CR and A1MCR (r = 0.85, P < .001). The cut-off values for UORM1 (2.53 mg/L) and UORM1CR (3.69 mg/g) for the early diagnosis of kidney impairment were obtained from receiver operating characteristic curves. UORM1CR obviously had higher diagnostic efficiency corresponding to 83.26% sensitivity and 90.32% specificity than UORM1. Likewise, its sensitivity was higher than those of ACR, A1MCR and eGFR. Bad glycaemic control had the highest risk of increased UORM1CR (odds ratio [OR] = 2.81, P < .001), while high HDL-C (high-density lipoprotein cholesterol) decreased the risk of increased UORM1CR (OR = 0.38, P = .017). CONCLUSION: The UORM1CR (>3.69 mg/g) has the high diagnostic efficiency for the early screening of renal impairment in type-2 diabetes patients. Furthermore, good glycaemic control and high HDL-C might be protective factors against UORM1CR increase.

STING positively regulates human ORMDL3 expression through TBK1-IRF3-STAT6 complex mediation.

Orosomucoid 1-like protein 3 (ORMDL3) is an asthma candidate gene associated with virus-triggered recurrent wheeze. Stimulator of interferon gene (STING) controls TLR-independent cytosolic responses to viruses. However, the association of STING with ORMDL3 is unclear. Here, we have shown that ORMDL3 expression shows a linear correlation with STING in recurrent wheeze patients. In elucidating the molecular mechanisms of the ORMDL3-STING relationship, we found that STING promoted the transcriptional activity of ORMDL3, which was significantly associated with increased levels of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6 (STAT6). Further study showed that via activation of TANK binding kinase 1 (TBK1), STING enhanced the phosphorylation and binding of IRF3 and STAT6, which upregulated ORMDL3 by binding to the promoter. Our results showed that STING positively regulated ORMDL3 through the TBK1-IRF3-STAT6 complex.

The Orosomucoid 1 protein is involved in the vitamin D - mediated macrophage de-activation process.

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

Association of the serological status of rheumatoid arthritis patients with two circulating protein biomarkers: A useful tool for precision medicine strategies.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and presence of systemic autoantibodies, with a great clinical and molecular heterogeneity. Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are routinely used for the diagnosis of RA. However, additional serological markers are needed to improve the clinical management of this disease, allowing for better patient stratification and the desirable application of precision medicine strategies. In the present study, we investigated those systemic molecular changes that are associated with the RF and ACPA status of RA patients. To achieve this objective, we followed a proteomic biomarker pipeline from the discovery phase to validation. First, we performed an iTRAQ-based quantitative proteomic experiment on serum samples from the RA cohort of the Hospital of Santiago de Compostela (CHUS). In this discovery phase, serum samples from the CHUS cohort were pooled according to their RF/ACPA status. Shotgun analysis revealed that, in comparison with the double negative group (RF-/ACPA-), the abundance of 12 proteins was altered in the RF+/ACPA+ pool, 16 in the RF+/ACPA- pool and 10 in the RF-/ACPA+ pool. Vitamin D binding protein and haptoglobin were the unique proteins increased in all the comparisons. For the verification phase, 80 samples from the same cohort were analyzed individually. To this end, we developed a Multiple Reaction Monitoring (MRM) method that was employed in a comprehensive targeted analysis with the aim of verifying the results obtained in the discovery phase. Thirty-one peptides belonging to 12 proteins associated with RF and/or ACPA status were quantified by MRM. In a final validation phase, the serum levels of alpha-1-acid glycoprotein 1 (A1AG1), haptoglobin (HPT) and retinol-binding protein 4 (RET4) were measured by immunoassays in the RA cohort of the Hospital of A Coruña (HUAC). The increase of two of these putative biomarkers in the double seropositive group was validated in 260 patients from this cohort (p = 0.009 A1AG1; p = 0.003 HPT). The increased level of A1AG1 showed association with RF rather than ACPA (p = 0.023), whereas HPT showed association with ACPA rather than RF (p = 0.013). Altogether, this study has allowed a further classification of the RA seropositive patients into two novel clusters: RF+A1AG+ and ACPA+HPT+. The determination of A1AG1 and HPT in serum would provide novel information useful for RA patient stratification, which could facilitate the effective implementation of personalized medicine in routine clinical practice.

Evaluation of IFIT3 and ORM1 as Biomarkers for Discriminating Active Tuberculosis from Latent Infection.

OBJECTIVE: Current commercially available immunological tests cannot be used for discriminating active tuberculosis (TB) from latent TB infection. To evaluate the value of biomarker candidates in the diagnosis of active TB, this study aimed to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) between patients with active TB and individuals with latent TB infection by transcriptome sequencing. METHODS: The differentially expressed genes in unstimulated PBMCs and in Mycobacterium tuberculosis (Mtb) antigen-stimulated PBMCs from patients with active TB and individuals with latent TB infection were identified by transcriptome sequencing. Selected candidate genes were evaluated in cohorts consisting of 110 patients with TB, 30 individuals with latent TB infections, and 50 healthy controls by quantitative real-time RT-PCR. Receiver operating characteristic (ROC) curve analysis was performed to calculate the diagnostic value of the biomarker candidates. RESULTS: Among the differentially expressed genes in PBMCs without Mtb antigen stimulation, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) had the highest area under curve (AUC) value (0.918, 95% CI: 0.852-0.984, P<0.0001) in discriminating patients with active TB from individuals with latent TB infection, with a sensitivity of 91.86% and a specificity of 84.00%. In Mtb antigen-stimulated PBMCs, orosomucoid 1 (ORM1) had a high AUC value (0.833, 95% CI: 0.752-0.915, P<0.0001), with a sensitivity of 81.94% and a specificity of 70.00%. CONCLUSION: IFIT3 and ORM1 might be potential biomarkers for discriminating active TB from latent TB infection.

miR-362-3p Targets Orosomucoid 1 to Promote Cell Proliferation, Restrain Cell Apoptosis and Thereby Mitigate Hypoxia/Reoxygenation-Induced Cardiomyocytes Injury.

This study aimed to investigate the mechanism of how miR-362-3p/orosomucoid 1 (ORM1) involved in hypoxia/reoxygenation (H/R)-induced cardiomyocytes injury. Based on data obtained from Gene Expression Omnibus (GEO) database, we revealed that ORM1 was highly expressed and positively correlated with the expression of inflammatory factors (MAPK1, MAPK3, IL1B and CASP9). miR-362-3p was identified as an upstream regulatory miRNA of ORM1 and negatively modulated the mRNA and protein expression levels of ORM1 in H/R-injured cardiomyocytes. Moreover, we found that miR-362-3p was downregulated in cardiomyocytes injured by H/R. The promoting influence of miR-362-3p mimic on the proliferation and the inhibitory effect of miR-362-3p mimic on the apoptosis of H/R-stimulated cardiomyocytes were eliminated by overexpression of ORM1. Furthermore, miR-362-3p affected the expression of MAPK1, MAPK3, IL1B and CASP9 in H/R-injured cardiomyocytes through targeting ORM1. Our outcomes illustrated that miR-362-3p exhibited a protective influence on H/R-induced cardiomyocytes through targeting ORM1.

Comparison of orosomucoid-1 immunoexpression and angiogenesis between oral squamous cell carcinoma cases with different histological grades.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common malignancy in this region, and thus, further elucidation of its tumoral mechanisms is important. One of the main roles of the acute-phase protein orosomucoid-1 (ORM1) is the promotion of angiogenesis, which is key for tumor nutrition and growth. AIM: Our aim was to evaluate the immunohistochemical expression of ORM1 and the angiogenic activity indicated by microvascular density (MVD) in OSCC samples according to histological grade. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded sections from 45 OSCC cases were submitted to immunohistochemistry: 25 were well-differentiated OSCC, 18 were moderately differentiated OSCC and 2 were poorly differentiated OSCC. ORM1 staining was evaluated by a semiquantitative method, and CD34-positive blood vessels were quantified to calculate the MVD. The results were statically analyzed. RESULTS: All cases exhibited immunoexpression of ORM1 and CD34. However, no significant differences were found between the expression of both markers among the histological grades. In addition, the presence of ORM1 in inflammatory cells and in the extracellular matrix was detected in most cases. CONCLUSION: These results suggest that the induction of angiogenesis is not the main role of ORM1 in OSCC and may be associated with the regulation of the immune/inflammatory response or the transport of protumoral molecules, such as sialyl-Lewis X or phorbol esters, which requires confirmation in future studies.

Orosomucoid 1 promotes epirubicin resistance in breast cancer by upregulating the expression of matrix metalloproteinases 2 and 9.

Orosomucoid 1 (ORM1) has been shown to be upregulated in the serum of breast cancer patients; however, the expression and function of ORM1 in breast cancer remains unknown. We measured the expression of ORM1 in breast cancer tissues and cell lines using qRT-PCR. A colony formation assay was done to assess cell proliferation and Transwell and wound healing assays were performed to determine the migration and invasion capacity of the cells, respectively. In addition, a CCK-8 assay was used to measure epirubicin cytotoxicity and western blot assays were done to analyze the putative mechanisms of epirubicin sensitivity. We found that the expression of ORM1 was upregulated in breast cancer tissues and cell lines. The expression of ORM1 enhanced the proliferation and migration of the cell lines. In contrast, down-regulation of ORM1 inhibited the expression of MMP-2 and MMP-9 and activation of the AKT/ERK signaling pathway. Therefore, ORM1 may represent a potential therapeutic target for breast cancer and promote epirubicin resistance by regulating the expression of MMP-2 and MMP-9, as well as activating the AKT/ERK signaling pathway.

Investigating the Association of Orosomucoid 1-like 3 (ORMDL3) Gene Polymorphism (rs12603332) with Susceptibility to Allergic Asthma in Iranian Northwestern Azeri Population.

Orosomucoid 1-like 3 (ORMDL3) gene, located on chromosome 17q21, is an asthma candidate gene that encodes ORMDL3. This molecule has been reported to play a role in airway remodeling and bronchial hyper-responsiveness. In this study, we aimed to investigate the possible association of ORMDL3 single nucleotide polymorphism (SNP) (rs12603332) with susceptibility to allergic asthma in Iranian Northwestern Azeri population. 193 asthmatic patients and 185 normal individuals were included. Genomic DNA was extracted and genotyping was performed by standard restriction fragment length polymorphism-polymerase chain reaction RFLP-PCR method using BstUI restriction enzyme. Our results showed dominant presence of TC genotype and C allele in both patients (49.2% and 59.8%, respectively) and controls (48.6% and 60%, respectively). Frequency of genotypes and alleles showed no significant difference between two groups (p=0.994 and p=1.00, respectively). None of alleles could be defined as risk allele for allergic asthma (OR=0.99, 0.88-1.12, 95% CI). We failed to show significant association between ORMDL3 rs12603332 with predisposition to allergic asthma in Iranian Northwestern Azeri population. More studies with larger number of participants should be done to find more reliable results for such association.

Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes.

The acute phase protein orosomucoid-1 (Orm1) is mainly expressed by hepatocytes (HPCs) under stress conditions. However, its specific function is not fully understood. Here, we report a role of Orm1 as an executer of HPC proliferation. Increases in serum levels of Orm1 were observed in patients after surgical resection for liver cancer and in mice undergone partial hepatectomy (PH). Transcriptome study showed that Orm1 became the most abundant in HPCs isolated from regenerating mouse liver tissues after PH. Both in vitro and in vivo siRNA-induced knockdown of Orm1 suppressed proliferation of mouse regenerating HPCs and human hepatic cells. Microarray analysis in regenerating mouse livers revealed that the signaling pathways controlling chromatin replication, especially the minichromosome maintenance protein complex genes were uniformly down-regulated following Orm1 knockdown. These data suggest that Orm1 is induced in response to hepatic injury and executes liver regeneration by activating cell cycle progression in HPCs.

Mechanisms and roles by which IRF-3 mediates the regulation of ORMDL3 transcription in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infancy, which is a major risk factor for recurrent wheezing and asthma. Orosomucoid 1-like protein 3 (ORMDL3) has been reported to associate with virus-triggered recurrent wheezing and asthma in children. However, little is known about how ORMDL3 is involved into RSV infection. In this study, we showed that the mRNA expression of ORMDL3 is significantly increased in the peripheral blood lymphocytes of infants with RSV-induced bronchiolitis compared with uninfected controls, also increased in bronchial epithelial cells and lung fibroblasts following RSV infection in vitro. To investigate the underlying mechanisms of RSV-induced ORMDL3 expression, we performed in silico analysis of the binding sites of several transcription factors in the ORMDL3 promoter. The proximal interferon-regulatory factor-3 (IRF-3) binding site positively regulated ORMDL3 transcription following exposure to RSV, as determined through mutational analysis. Overexpression and RNA interference experiments targeting IRF-3 showed that it regulates the expression of ORMDL3 following RSV exposure. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay showed that IRF-3 binds directly to the promoter of the ORMDL3 gene. Furthermore, we confirmed that expression of IRF-3 is significantly increased and shows a strong linear correlation with increased ORMDL3 in the peripheral blood lymphocytes from infants with RSV-induced bronchiolitis. Our results indicate that IRF-3 is an important regulator of ORMDL3 induction following RSV infection by binding directly to the promoter of ORMDL3, which may be implicated in the inflammatory and immune reactions involved in bronchiolitis and wheezing diseases.

Orosomucoid-1 Expression in Ameloblastoma Variants.

Odontogenic tumors constitute a group of heterogeneous lesions of benign and malignant neoplasms with variable aggressiveness. Ameloblastomas are a group of benign but locally invasive neoplasms that occur in the jaws and are derived from epithelial elements of the tooth-forming apparatus. We previously described orosomucoid-1 protein expression in odontogenic myxomas. However, whether orosomucoid-1 is expressed in other odontogenic tumors remains unknown. Since orosomucoid-1 belongs to a group of acute-phase proteins and has many functions in health and disease, we identified and analyzed orosomucoid-1 expression in ameloblastoma variants and ameloblastic carcinoma using western blot and immunohistochemical techniques. Thirty cases of ameloblastoma were analyzed for orsomucoid-1; five specimens were fresh for western blot study (four benign ameloblastomas and one ameloblastic carcinoma), and 25 cases of benign ameloblastoma for immunohistochemical assays. Orosomucoid-1 was widely expressed in each tumor variant analyzed in this study, and differential orosomucoid-1 expression was observed between benign and malignant tumor. Orosomucoid-1 may play an important role in the behavior of ameloblastomas and influence the biology and development of the variants of this tumor.

The increased excretion of urinary orosomucoid 1 as a useful biomarker for bladder cancer.

Improving the early detection rate and prediction of bladder cancer remains a great challenge in management of this disease. To examine the value of urinary orosomucoid 1 (ORM1) for the early detection and surveillance of bladder cancer, two-dimensional differential gel electrophoresis (2-DE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOFMS) were applied to identify the differently expressed proteins in urine between bladder cancer and healthy controls. Thirteen different proteins including ORM1 were identified. After verification by western blotting, the ORM1 expressions were quantified in 186 urine samples by enzyme-linked immunosorbent assay (ELISA) correcting for creatinine expression. ELISA quantification showed the urinary ORM1-Cr was found to be higher in bladder cancer patients compared to controls and benign cases (7172.23±3049.67 versus 2243.16±969.01, 2493.48±830.37 ng/ml, respectively, P<0.0001). Furthermore, the pearson correlation analysis indicated that urinary ORM1 had high positive correlation with the pathology classification of bladder cancer. Receiver operating characteristic (ROC) analysis was used to calculate the cut-off value for early diagnosis of bladder cancer, and rendered an optimum cut-off value of 3912.97 ng/mg corresponding to 91.96% sensitivity and 94.34% specificity. Moreover, a cut-off value with 7351.28 ng/mg was utilized to distinguish infiltrating urothelial carcinoma from bladder cancer patients corresponding to 91.89% sensitivity and 90.67% specificity. In conclusion, our findings suggested the elevated urinary ORM1 could be a useful biomarker for bladder cancer. Further research is warranted to elucidate the pathogenic mechanisms of elevated ORM1.

E3 ubiquitin ligase Cbl-b suppresses human ORMDL3 expression through STAT6 mediation.

Orosomucoid 1-Like Protein 3 (ORMDL3) is an asthma candidate gene and Casitas B lineage lymphoma b (Cbl-b), an E3 ubiquitin ligase, is a critical factor in maintaining airway immune tolerance. However, the association of Cbl-b with ORMDL3 for asthma is unclear. Here, we show that expression of ORMDL3 is significantly increased and shows a strong linear correlation with decreased Cbl-b in the peripheral blood of recurrent wheeze patients. To elucidate the molecular mechanisms underlying this correlation, we identified that Cbl-b suppressed the transcriptional activity and mRNA expression of ORMDL3 in vivo. Further investigation showed that phosphorylation of signal transducer and activator of transcription 6 (STAT6) was induced by interleukin 4 bound to the ORMDL3 promoter, while Cbl-b reduced the phosphorylation of STAT6. Our results show that Cbl-b suppresses human ORMDL3 expression through STAT6.