Serum mRNA levels of cytokeratin-19 and vascular endothelial growth factor in oral squamous cell carcinoma and oral potentially malignant disorders using RT-PCR.

BACKGROUND: Oral cancers, which include tumors of the oral cavity, salivary glands, and pharynx, are becoming increasingly prevalent worldwide. Squamous cell carcinoma accounts for over 90% of malignant oral lesions, with oral squamous cell carcinoma (OSCC) being notably common in the Indian subcontinent and other regions of Asia. This is especially true in South-Central Asia, including Sri Lanka, where it is particularly prevalent among men. This study aims to evaluate the levels of Vascular Endothelial Growth Factor-A (VEGF-A) and Cytokeratin-19 (CK-19) mRNAs in whole blood as a potential method for the early detection of OSCC. METHODS: The study included 40 patients (each from OSCC, Oral Submucous Fibrosis (OSF), Oral Leukoplakia (OLK), Oral Lichen Planus (OLP), and 10 healthy controls. The expression levels of VEGF-A and CK-19 mRNAs were measured from extracellular RNA extracted from whole blood samples using real-time reverse transcription polymerase chain reaction (RT-PCR) with sequence-specific primers. Receiver operating characteristic (ROC) curve analysis was used to evaluate the effectiveness of these biomarkers in detecting OSCC. RESULTS: The results demonstrated a significant increase in blood transcripts of the candidate mRNAs CK-19 and VEGF-A in patients with OSCC, OSF, OLK, and OLP. The Wilcoxon signed-rank test revealed a p-value of 0.002 for each specific comparison between diseased patients and healthy controls (i.e., OSCC vs. HC, OSF vs. HC, OLP vs. HC, OLK vs. HC) for both CK-19 and VEGF-A. When these two biomarkers were used together, they provided a 60% predictive probability for patients with OSCC (p = 0.023). CONCLUSION: This study highlights the efficacy of blood mRNA transcriptome diagnostics in detecting OSCC. This innovative clinical approach has the potential to be a robust, efficient, and reliable tool for early cancer detection. Blood-based transcriptomes could be further explored for their effectiveness in various health contexts and for routine health monitoring.

Simultaneous Detection of Collagen I Alpha II and Cytokeratin 19 mRNA by Multiplex qPCR in Liquid Biopsy in Diagnosis of Patients with Resectable Solid Tumors.

The early detection of tumors is one of the key factors in increasing overall survival in cancer patients. A wide range of cancers still do not have a system of early diagnosis; therefore, the development of new non-invasive tools in this line is essential. Accordingly, the objective of our work was to develop a non-invasive screening method for the early detection of various carcinomas in plasma using a panel that combines two markers using RT-qPCR. A retrospective case-control study was conducted to develop a cancer screening test based on the detection of stromal and epithelial biomarkers (COL1A2 and KRT19) in plasma. The expression of biomarkers was evaluated using multiplex quantitative PCR applied to 47 cases with non-metastatic tumors and 13 control participants. For both biomarkers, a cut-off value was stablished using Youden's J index through ROC curve analysis and areas under the curve (AUC) were calculated. The plasma mRNA expression level of both biomarkers was significantly higher in diseased versus healthy patients. Moreover, ROC curve analysis showed an AUC value of 0.897 for the combined model. This model also resulted in a cutoff value of 0.664, as well as a sensitivity of 83% and a specificity of 84.6%. These results suggest that the plasma expression levels of COL1A2 and KRT19 could a have potential role in detecting various types of cancer at the early stages. The combined analysis of both stromal and epithelial biomarkers would provide a non-invasive screening method that would allow us to differentiate patients with an active neoplastic process.

Expression and significance of CEMIP and CYP11B2 in serum in women with foetal growth restriction.

BACKGROUND: Foetal growth restriction (FGR) occurs when a foetus fails to reach its growth potential. This observational study assessed the expression and significance of cell migration-including protein (CEMIP) and aldosterone synthase (CYP11B2) in the serum of pregnant women with FGR. METHODS: 40 singleton FGR-suffered pregnant women, as well as 40 normal singleton pregnant women, were enrolled. The expression of CEMIP and CYP11B2 in serum was detected in early pregnancy. The correlations between parameters were evaluated. The predictive variables for FGR were determined. The diagnostic value of CEMIP and CYP11B2 for FGR was analysed. RESULTS: CEMIP and CYP11B2 mRNA expression in the serum of pregnant women with FGR decreased (both P < 0.001). CEMIP (95%CI: 0.802-0.921, P < 0.001) and CYP11B2 (95%CI: 0.795-0.907, P < 0.001) mRNA expression in serum and soluble fms like tyrosine kinase-1 (sFLT1)/placental growth factor (PlGF) ratio (95%CI: 0.866-0.974, P < 0.001) were independent predictors of FGR, and CEMIP (r = -0.578, P = 0.001) and CYP11B2 (r = -0.602, P < 0.001) mRNA expression in serum were negatively correlated with sFLT1/PlGF ratio. CEMIP (AUC = 0.741) and CYP11B2 (AUC = 0.764) mRNA expression in serum had good diagnostic value for FGR. CONCLUSION: The expression of CEMIP and CYP11B2 is reduced in the serum of pregnant women with FGR and may become new diagnostic markers for FGR.

Foetal growth restriction is the inability of the foetus to reach its growth potential in the uterus due to various factors. This study aimed to investigate the expression and significance of cell migration-including protein and aldosterone synthase in serum of pregnant women with foetal growth restriction. In our study, we found that the expression of cell migration-including protein and aldosterone synthase in serum of pregnant women with foetal growth restriction were decreased. Cell migration-including protein and aldosterone synthase expression was negatively correlated with soluble fms like tyrosine kinase-1/placental growth factor ratio. In addition, the study also found that cell migration-including protein and aldosterone synthase expression in serum had good diagnostic value for foetal growth restriction.

Diagnostic and Prognostic Value of Methyltransferase Like 13 in Hepatocellular Carcinoma Based on Bioinformatics Analysis.

BACKGROUND/AIMS:  Hepatocellular carcinoma (HCC) is one of the cancers with the highest incidence and mortality rates. This study aims to explore the diagnostic and prognostic utility of methyltransferase like 13 (METTL13) in patients with HCC via bioinformatics analysis. MATERIALS AND METHODS:  We obtained mRNA data of HCC from the database of the Cancer Genome Atlas (TCGA), drawing survival curve by R 4.2.1 software. Cox regression analysis was conducted based on tumor stage and METTL13 expression. The GSE114564 dataset was chosen from the Gene Expression Omnibus. The differences in serum METTL13 levels between the groups of early HCC (eHCC) and non-cancer controls were evaluated. Using a receiver operating characteristic curve, we calculated the area under the curve (AUC) of serum METTL13 for diagnosing eHCC. RESULTS:  A total of 225 cases with HCC were screened from TCGA, and 29 cases were normal controls. The results showed that the METTL13 expression in the HCC group was higher than that in the normal controls (P <.001). The univariate [hazard ratio (HR) = 1.895, P = .006] and multivariate Cox regression (HR = 1.702, P = .037) analyses showed that high METTL13 expression reduced overall survival in HCC. Serum METTL13 levels were higher in the eHCC group than in the non-cancer controls (P = .008). The optimum AUC for predicting eHCC by serum METTL13 was 0.7091. CONCLUSION:  Serum METTL13 has a moderate diagnostic value for eHCC. High METTL13 expression is correlated with a worse prognosis in patients with HCC. Methyltransferase like 13 possesses the potential to be a novel biomarker for HCC.

Bacterial sepsis causes more dramatic pathogenetic changes in the Th1 pathway than does viral (COVID-19) sepsis: a prospective observational study of whole blood transcriptomes.

OBJECTIVES: This study aimed to comprehensively compare host responses of patients with bacterial sepsis and those with viral (COVID-19) sepsis by analyzing messenger RNA (mRNA) and microRNA (miRNA) profiles to shed light on their distinct pathophysiological mechanisms. DESIGN: Prospective observational study. SETTING: Whole blood RNA sequencing was used to analyze mRNA and miRNA profiles of patients diagnosed as having bacterial sepsis or viral (COVID-19) sepsis at the Department of Trauma and Emergency Medicine, Osaka University Graduate School of Medicine. PATIENTS: Twenty-two bacterial sepsis patients, 35 viral (COVID-19) sepsis patients, and 15 healthy subjects admitted to the department were included. We diagnosed bacterial sepsis patients according to the sepsis-3 criterion that the Sequential Organ Failure Assessment score must increase to 2 points or more among patients with suspected infections. Viral (COVID-19) sepsis patients were diagnosed using SARS-CoV-2 RT-PCR testing, and presence of pneumonia was assessed through chest computed tomography scans. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: For RNA sequencing, 14,500 mRNAs, 1121 miRNAs, and 2556 miRNA-targeted mRNAs were available for analysis in the bacterial sepsis patients. Numbers of genes showing upregulated: downregulated gene expression (false discovery rate < 0.05, |log2 fold change| > 1.5) were 256:2887 for mRNA, 53:5 for miRNA, and 49:2507 for miRNA-targeted mRNA. Similarly, in viral (COVID-19) sepsis patients, 14,500 mRNAs, 1121 miRNAs, and 327 miRNA-targeted mRNAs were analyzed, with numbers of genes exhibiting upregulated: downregulated gene expression of 672:1147 for mRNA, 3:4 for miRNA, and 165:162 for miRNA-targeted mRNA. This analysis revealed significant differences in the numbers of upregulated and downregulated genes expressed and pathways between the bacterial sepsis and viral (COVID-19) sepsis patients. Bacterial sepsis patients showed activation of the PD-1 and PD-L1 cancer immunotherapy signaling pathway and concurrent suppression of Th1 signaling. CONCLUSION: Our study illuminated distinct molecular variances between bacterial sepsis and viral (COVID-19) sepsis. Bacterial sepsis patients had a greater number of upregulated and downregulated genes and pathways compared to viral (COVID-19) sepsis patients. Especially, bacterial sepsis caused more dramatic pathogenetic changes in the Th1 pathway than did viral (COVID-19) sepsis.

Integration and validation of host transcript signatures, including a novel 3-transcript tuberculosis signature, to enable one-step multiclass diagnosis of childhood febrile disease.

BACKGROUND: Whole blood host transcript signatures show great potential for diagnosis of infectious and inflammatory illness, with most published signatures performing binary classification tasks. Barriers to clinical implementation include validation studies, and development of strategies that enable simultaneous, multiclass diagnosis of febrile illness based on gene expression. METHODS: We validated five distinct diagnostic signatures for paediatric infectious diseases in parallel using a single NanoString nCounter® experiment. We included a novel 3-transcript signature for childhood tuberculosis, and four published signatures which differentiate bacterial infection, viral infection, or Kawasaki disease from other febrile illnesses. Signature performance was assessed using receiver operating characteristic curve statistics. We also explored conceptual frameworks for multiclass diagnostic signatures, including additional transcripts found to be significantly differentially expressed in previous studies. Relaxed, regularised logistic regression models were used to derive two novel multiclass signatures: a mixed One-vs-All model (MOVA), running multiple binomial models in parallel, and a full-multiclass model. In-sample performance of these models was compared using radar-plots and confusion matrix statistics. RESULTS: Samples from 91 children were included in the study: 23 bacterial infections (DB), 20 viral infections (DV), 14 Kawasaki disease (KD), 18 tuberculosis disease (TB), and 16 healthy controls. The five signatures tested demonstrated cross-platform performance similar to their primary discovery-validation cohorts. The signatures could differentiate: KD from other diseases with area under ROC curve (AUC) of 0.897 [95% confidence interval: 0.822-0.972]; DB from DV with AUC of 0.825 [0.691-0.959] (signature-1) and 0.867 [0.753-0.982] (signature-2); TB from other diseases with AUC of 0.882 [0.787-0.977] (novel signature); TB from healthy children with AUC of 0.910 [0.808-1.000]. Application of signatures outside of their designed context reduced performance. In-sample error rates for the multiclass models were 13.3% for the MOVA model and 0.0% for the full-multiclass model. The MOVA model misclassified DB cases most frequently (18.7%) and TB cases least (2.7%). CONCLUSIONS: Our study demonstrates the feasibility of NanoString technology for cross-platform validation of multiple transcriptomic signatures in parallel. This external cohort validated performance of all five signatures, including a novel sparse TB signature. Two exploratory multi-class models showed high potential accuracy across four distinct diagnostic groups.

Functional analysis of a panel of molecular markers for diagnosis of systemic lupus erythematosus in rats.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a diverse autoimmune disease that arises from a combination of complex genetic factors and environmental influences. While circRNAs and miRNAs have recently been identified as promising biomarkers for disease diagnosis, their specific expression patterns, and clinical implications in SLE are not yet fully understood. AIM OF THE WORK: The aim of the present study was to determine the role of a panel of noncoding-RNAs specifically circRNAs (circ-TubD1, circ-CDC27, and circ-Med14), along with miRNA (rno-miR-146a-5p) and mRNA (TRAF6), as novel minimally invasive diagnostic biomarkers for experimentally induced SLE. Additionally, the study involved an insilico bioinformatics analysis to explore potential pathways involved in the pathogenesis of SLE, aiming to enhance our understanding of the disease, enable early diagnosis, and facilitate improved treatment strategies. MATERIALS AND METHODS: SLE was induced in rats using single IP injection of incomplete Freund's adjuvant (IFA). The Induction was confirmed by assessing the ANA and anti-ds DNA levels using ELSA technique. qPCR analysis was conducted to assess the expression of selected RNAs in sera collected from a group of 10 rats with induced SLE and a control group of 10 rats. In addition, bioinformatics and functional analysis were used to construct a circRNA-miRNA-mRNA network and to determine the potential function of these differentially expressed circRNAs. RESULTS: SLE rats demonstrated significantly higher expression levels of circ-CDC27, circ-Med14, and rno-miR-146a-5p as well as TRAF6, with lower expression level of circ-TubD1 in sera of SLE rats relative to controls. ROC curve analysis indicated that all the selected non-coding RNAs could serve as potential early diagnostic markers for SLE. In addition, the expression level of circ-TubD1 was negatively correlated with rno-miR-146a-5p, however, rno-miR-146a-5p was positively correlated with TRAF6. Bioinformatic analysis revealed the incorporation of the circRNAs targeted genes in various immune system and neurodegeneration pathways. CONCLUSIONS: Therefore, circRNAs; circ-TubD1, circ-CDC27, and circ-Med14, in addition to the miRNA (rno-miR-146a-5p) and mRNA (TRAF6) may be involved in the development of SLE and may have promising roles for future diagnosis and targeted therapy.

Multicenter Validation of lncRNA and Target mRNA Diagnostic and Prognostic Biomarkers of Acute Ischemic Stroke From Peripheral Blood Leukocytes.

BACKGROUND: Long noncoding RNA (lncRNA) and mRNA profiles in leukocytes have shown potential as biomarkers for acute ischemic stroke (AIS). This study aimed to identify altered lncRNA and target mRNA profiles in peripheral blood leukocytes as biomarkers and to assess the diagnostic value and association with AIS prognosis. METHODS AND RESULTS: Differentially expressed lncRNAs (DElncRNAs) and differentially expressed target mRNAs (DEmRNAs) were screened by RNA sequencing in the discovery set, which consisted of 10 patients with AIS and 20 controls. Validation sets consisted of a multicenter (311 AIS versus 303 controls) and a nested case-control study (351 AIS versus 352 controls). The discriminative value of DElncRNAs and DEmRNAs added to the traditional risk factors was estimated with the area under the curve. NAMPT-AS, FARP1-AS1, FTH1, and NAMPT were identified in the multicenter case-control study (P<0.05). LncRNA NAMPT-AS was associated with cis-target mRNA NAMPT and trans-target mRNA FTH1 in all validation sets (P<0.001). Similarly, AIS cases exhibited upregulated lncRNA FARP-AS1 and FTH1 expression (P<0.001) in the nested case-control study (P<0.001). Furthermore, lncRNA FARP1-AS1 expression was upregulated in AIS patients at discharge with an unfavorable outcome (P<0.001). Positive correlations were found between NAMPT expression level and NIHSS scores of AIS patients (P<0.05). Adding 2 lncRNAs and 2 target mRNAs to the traditional risk factor model improved area under the curve by 22.8% and 5.2% in the multicenter and the nested case-control studies, respectively. CONCLUSIONS: lncRNA NAMPT-AS and FARP1-AS1 have potential as diagnostic biomarkers for AIS and exhibit good performance when combined with target mRNA NAMPT and FTH1.

Evaluation of a blood miRNA/mRNA signature to follow-up Lu-PRRT therapy for G1/G2 intestinal neuroendocrine tumors.

Background: 177Lu-oxodotreotide peptide receptor therapy (LuPRRT) is an efficient treatment for midgut neuroendocrine tumors (NETs) of variable radiological response. Several clinical, biological, and imaging parameters may be used to establish a relative disease prognosis but none is able to predict early efficacy or toxicities. We investigated expression levels for mRNA and miRNA involved in radiosensitivity and tumor progression searching for correlations related to patient outcome during LuPRRT therapy. Methods: Thirty-five patients received LuPRRT for G1/G2 midgut NETs between May 2019 and September 2021. Peripheral blood samples were collected prior to irradiation, before and 48 h after the second and the fourth LuPRRT, and at 6-month follow-up. Multiple regression analyses and Pearson correlations were performed to identify the miRNA/mRNA signature that will best predict response to LuPRRT. Results: Focusing on four mRNAs and three miRNAs, we identified a miRNA/mRNA signature enabling the early identification of responders to LuPRRT with significant reduced miRNA/mRNA expression after the first LuPRRT administration for patients with progressive disease at 1 year (p < 0.001). The relevance of this signature was reinforced by studying its evolution up to 6 months post-LuPRRT. Moreover, nadir absolute lymphocyte count within the first 2 months after the first LuPRRT administration was significantly related to low miRNA/mRNA expression level (p < 0.05) for patients with progressive disease. Conclusion: We present a pilot study exploring a miRNA/mRNA signature that correlates with early hematologic toxicity and therapeutic response 12 months following LuPRRT. This signature will be tested prospectively in a larger series of patients.

I-FABP protein/mRNA and IL-6 as biomarkers of intestinal barrier dysfunction in neonates with necrotizing enterocolitis and SPF BALB/c mouse models.

OBJECTIVE: Neonatal necrotizing enterocolitis (NEC) is a serious intestinal inflammatory disease. We investigated intestinal fatty acid binding protein (I-FABP), I-FABP mRNA, and interleukin-6 (IL-6) as potential diagnostic biomarkers in NEC. METHODS: Forty mice were subjected to hypoxic-ischemic intestinal injury, and then serum I-FABP protein and mRNA levels were quantified. Ileal tissue pathological scores were determined by hematoxylin and eosin staining. I-FABP expression levels and translocation in these tissues were detected using western blotting and immunofluorescence, respectively. Samples from 30 human neonates with NEC and 30 healthy neonates had serum I-FABP protein/mRNA and IL-6 levels measured. RESULTS: The mouse ileal tissue pathological score and I-FABP levels, as well as serum I-FABP and I-FABP mRNA levels, were significantly higher in the model group than in the control group. Serum I-FABP, I-FABP mRNA, and IL-6 levels were significantly higher in human neonates with NEC than in the healthy group. Logistic regression and receiver operating curve analyses revealed that I-FABP protein/mRNA and IL-6 levels could be diagnostic biomarkers for NEC. CONCLUSIONS: I-FABP protein/mRNA and IL-6 levels are useful biomarkers of intestinal ischemic injury in neonates with NEC. The combined detection of I-FABP protein/mRNA and IL-6 is recommended rather than using a single biomarker.

Comparison of serum levels of IL-10 and IL-11 and mRNA expression of IL-10, IL-11, COX-2, BCL6, and ZEB Family in peripheral blood mononuclear cells (PBMC) of women with polycystic ovary syndrome and healthy individuals.

BACKGROUND: The roles of IL-10, IL-11, COX-2, BCL6, ZEB1, and ZEB2 genes in the potential correlation between polycystic ovary syndrome (PCOS), inflammation, and cancer remain controversial. AIMS: This study aimed to compare serum levels of IL-10 and IL-11 and gene expression of IL-10, IL-11, COX-2, BCL6, ZEB1, and ZEB2 in PBMCs of women with PCOS and healthy controls. METHODS: A case-control study included 40 women with PCOS as the case group and 40 healthy women as controls. Group matching for age and BMI was performed. Serum levels of IL-10 and IL-11 were assessed using ELISA, while gene expression was measured using real-time PCR. Parameters were compared between groups, and correlations among gene expression and serum levels were explored. RESULTS: In comparison to healthy women, women with PCOS exhibited a significant decrease in the expression of COX-2 and IL-10 genes (p<0.001), alongside a significant increase in ZEB2 gene expression (p<0.001). There were no significant differences observed in the expression of IL-11, BCL6, and ZEB1 genes. Furthermore, the serum level of IL-10 was significantly lower in women with PCOS compared to the control group (p<0.001), while no significant difference was found in IL-11 levels. Additionally, no significant correlations were identified between gene expression and serum levels. CONCLUSION: In women with PCOS, reduced IL-10 gene expression may indicate inflammation and serve as a diagnostic biomarker. However, conflicting findings on COX-2 expression complicate understanding. Elevated ZEB2 expression in PCOS women may lead to infertility, epithelial-mesenchymal transition, and aggressive phenotypes.

Ischemic Stroke with Comorbid Cancer Has Specific miRNA-mRNA Networks in Blood That Vary by Ischemic Stroke Mechanism.

OBJECTIVE: Approximately half of ischemic strokes (IS) in cancer patients are cryptogenic, with many presumed cardioembolic. We evaluated whether there were specific miRNA and mRNA transcriptome architectures in peripheral blood of IS patients with and without comorbid cancer, and between cardioembolic versus noncardioembolic IS etiologies in comorbid cancer. METHODS: We studied patients with cancer and IS (CS; n = 42), stroke only (SO; n = 41), and cancer only (n = 28), and vascular risk factor-matched controls (n = 30). mRNA-Seq and miRNA-Seq data, analyzed with linear regression models, identified differentially expressed genes in CS versus SO and in cardioembolic versus noncardioembolic CS, and miRNA-mRNA regulatory pairs. Network-level analyses identified stroke etiology-specific responses in CS. RESULTS: A total of 2,085 mRNAs and 31 miRNAs were differentially expressed between CS and SO. In CS, 122 and 35 miRNA-mRNA regulatory pairs, and 5 and 3 coexpressed gene modules, were associated with cardioembolic and noncardioembolic CS, respectively. Complement, growth factor, and immune/inflammatory pathways showed differences between IS etiologies in CS. A 15-gene biomarker panel assembled from a derivation cohort (n = 50) correctly classified 81% of CS and 71% of SO participants in a validation cohort (n = 33). Another 15-gene panel correctly identified etiologies for 13 of 13 CS-cardioembolic and 11 of 11 CS-noncardioembolic participants upon cross-validation; 11 of 16 CS-cryptogenic participants were predicted cardioembolic. INTERPRETATION: We discovered unique mRNA and miRNA transcriptome architecture in CS and SO, and in CS with different IS etiologies. Cardioembolic and noncardioembolic etiologies in CS showed unique coexpression networks and potential master regulators. These may help distinguish CS from SO and identify IS etiology in cryptogenic CS patients. ANN NEUROL 2024;96:565-581.

Relationship between circulating thrombospondin-1 messenger ribonucleic acid and microribonucleic acid-194 levels in Chinese patients with type 2 diabetic kidney disease: The outcomes of a case-control study.

AIMS/INTRODUCTION: We investigated the relationship of circulating TSP-1 mRNA and miR-194 with diabetic kidney disease's degree. MATERIALS AND METHODS: We enrolled 167 hospitalized type 2 diabetes patients in the endocrinology department. Patients were split into three groups according to urinary microalbumin: A, B and C. The control group comprised healthy outpatients (n = 163). The quantities of microribonucleic acid (miR)-194 and thrombospondin-1 (TSP-1) messenger ribonucleic acid (mRNA) in the participants' circulation were measured using a quantitative real-time polymerase chain reaction. RESULTS: Circulating TSP-1 mRNA (P = 0.024) and miR-194 (P = 0.029) expressions significantly increased in type 2 diabetes patients. Circulating TSP-1 mRNA (P = 0.040) and miR-194 (P = 0.007) expression levels differed significantly among the three groups; circulating TSP-1 mRNA expression increased with urinary microalbumin. However, miR-194 declined in group B and increased in group C. Circulating TSP-1 mRNA was positively correlated with cystatin-c (r = 0.281; P = 0.021) and microalbumin/creatinine ratio (UmALB/Cr; r = 0.317; P = 0.009); miR-194 was positively correlated with UmALB/Cr (r = 0.405; P = 0.003). Stepwise multivariate linear regression analysis showed cystatin-c (ß = 0.578; P = 0.021) and UmALB/Cr (ß = 0.001; P = 0.009) as independent factors for TSP-1 mRNA; UmALB/Cr (ß = 0.005; P = 0.028) as an independent factor for miR194. Areas under the curve for circulating TSP-1 mRNA and miR194 were 0.756 (95% confidence interval 0.620-0.893; sensitivity 0.69 and specificity 0.71, P < 0.01) and 0.584 (95% confidence interval 0.421-0.748; sensitivity 0.54 and specificity 0.52, P < 0.01), respectively. CONCLUSIONS: Circulating TSP-1 mRNA and miR-194 expressions significantly increased in type 2 diabetes patients. The microalbumin group had lower levels of miR-194 (a risk factor that is valuable for type 2 diabetes kidney disease evaluation).

The Impact of the IL-10 Gene Polymorphism on mRNA Expression and IL-10 Serum Concentration in Polish Lupus Patients.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of autoantibodies against a lot of nuclear components. Despite many studies on the genetic background of this disease, the pathogenesis remains unclear. The aim of the study is to comprehensively evaluate the polymorphism of the IL-10 promoter gene, its mRNA expression, and the serum IL-10 concentration of SLE female patients and females age-matched controls. Analyzing the association between the level of the tested cytokine and the polymorphism genotype-1082; -819; -592, we found statistically higher serum IL-10 levels in SLE patients compared to in healthy controls (11.9 ± 2.2 pg/mL vs. 9.4 ± 1.7 pg/mL, accordingly; p < 0.0001). We did not find statistically significant differences in the gene polymorphism of IL-10 among SLE patients and controls. The most significant observation derived from our study is that IL-10 mRNA transcripts are upregulated in SLE patients compared to in healthy controls (p < 0.0001). According to our results, the presence of the IL-10 genetic polymorphism has no clinical significance for the development of SLE, and subsequent differences in mRNA and IL-10 concentration results from the influence of other factors which should be the subject of further research.

Correlation of Blood and Intestinal Mucosa miR-34a Expressions with Disease Severity in Ulcerative Colitis Patients.

BACKGROUND: miR-34a has been implicated in many autoimmune diseases and gastrointestinal diseases. However, the expression of miR-34 in ulcerative colitis (UC) patients were not fully studied. This study was performed to in-vestigate the association of blood and intestinal tissue miR-34a expression of patients with disease severity in UC patients. METHODS: Our study enrolled 82 patients with UC and 80 age- and gender- matched healthy individuals. Blood miR-34a expressions were detected using reverse transcription-polymerase chain reaction (RT-PCR). Local intestinal miR-34a, STAT3 mRNA and IL-23 mRNA expressions were also detected in the lesioned area and adjacent non-affected intestinal tissue in patients. Disease severity of UC was assessed by Mayo score. The diagnostic value of both blood and local miR-34a expression for UC patients was assessed by receiver operating characteristic (ROC) curve. RESULTS: Blood miR-34a was increased in UC patients in contrast with healthy individuals with statistical significance. In UC patients, local intestinal miR-34a expressions were markedly upregulated compared to adjacent non-affected intestinal tissue. Local intestinal miR-34a expressions were positively correlated with STAT3 mRNA and IL-23 mNRA. Both blood and local miR-34a expressions were significantly and positively related to Mayo scores. ROC curve analysis indicated that both blood and local miR-34a expressions may act as decent marker for Mayo grade. CONCLUSIONS: Blood and intestinal tissue miR-34a expressions are correlated with disease severity in UC patients. Both blood and intestinal tissue miR-34a expressions may serve as potential diagnostic and prognostic makers for UC. Therapeutic methods targeting miR-34a may act as potential ways for UC treatment.

Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

Serum NfL and EGFR/NfL ratio mRNAs as biomarkers for phenotype and disease severity of myelin oligodendrocyte glycoprotein IgG-associated disease.

Background and purpose: Myelin oligodendrocyte glycoprotein (MOG) IgG is frequently elevated in pediatric patients with acquired demyelinating syndrome (ADS). However, no specific biomarkers exist for phenotype classification, symptom severity, prognosis, and treatment guidance of MOG-IgG-associated disease (MOGAD). This study evaluated neurofilament light chain (NfL) and endothelial growth factor receptor (EGFR) mRNA expression levels in serum and cerebrospinal fluid (CSF) as potential biomarkers for MOGAD in Chinese children. Methods: This was a cross-sectional and single-center study. We enrolled 22 consecutive pediatric patients hospitalized with MOGAD and 20 control pediatric patients hospitalized for noninflammatory neurological diseases in Hebei Children's Hospital. Serum and CSF were collected from MOGAD patients within 3 days before immunotherapy. The mRNA levels of NfL and EGFR in serum and CSF were measured by real-time polymerase chain reaction (qPCR), and the EGFR/NfL ratio mRNA was calculated. These measurement values were then compared between disease groups and among MOGAD phenotypes. In addition, the correlations between the mRNAs of three markers (NfL, EGFR, EGFR/NfL ratio), extended disability status scale (EDSS) scores, and clinical phenotypes were analyzed. Results: Serum and CSF NfL mRNA levels were significantly higher of acute-stage MOGAD patients than those of control patients (p< 0.05 and p< 0.01, respectively), while the mRNA levels of serum EGFR and EGFR/NfL ratio were significantly lower of MOGAD patients than those of controls (p < 0.05, p < 0.0001). Serum NfL mRNA was significantly correlated with mRNA of serum EGFR (r =0.480, p < 0.05). Serum and CSF NfL mRNA levels in MOGAD patients with the ADEM-like phenotype were also significantly higher than those in control patients (p < 0.01, p < 0.01) and optic neuritis (ON) phenotype (p < 0.05, p < 0.05). Both mRNAs of NfL in CSF and EGFR/NfL ratio in serum were correlated with EDSS scores (p < 0.05, r = 0.424; p < 0.05, r= -0.521). Conclusion: The mRNA levels of elevated NfL in serum and CSF as well as lower EGFR and EGFR/NfL ratio in serum could help distinguish acute-phase MOGAD. Higher mRNA levels of NfL in serum and CSF of MOGAD patients help distinguish ADEM-like phenotype. In addition, serum EGFR/NfL mRNA ratio is indicative of disease severity in pediatric patients with MOGAD. Further investigations are warranted to elucidate the pathological mechanisms underlying these associations.

Prediction of preeclampsia using maternal circulating mRNAs in early pregnancy.

PURPOSE: Preeclampsia (PE) is one of the most common and serious complications of pregnancy, and novel methods for the early prediction of PE are needed for clinical application. METHODS: In this study, a circulating cell-free RNA (cfRNA) panel of target genes for PE prediction was designed and validated in a case-control cohort and a nested case-control cohort. The QPCR was applied to quantify the copy number of cfRNA, and the data were normalized as multiples of the median. Ratios of serum placental growth factor (PIGF) and soluble fms-like tyrosine kinase 1 (sFLT-1) were also measured, and transabdominal ultrasonography was conducted for subjects in the prospective cohort. Binary logistic regression models for PE prediction were constructed and tested. RESULTS: Our results revealed that the women with PE showed significant alterations in serum cfRNA profiles from early pregnancy onward and before the onset of PE symptoms. Compared with PIGF/sFLT-1 measurement and ultrasonographic imaging, cfRNA test can detect PE at a very early stage of pregnancy. The predictive model exhibited the best performance at gestation week 32, with a detection rate of 100%. At 12 weeks of gestation, the model still manifested an area under curve (AUC) of 0.9144, and sensitivity of 1.0000. If combined with clinical parameters and ultrasonographic indicators, the model can achieve the highest AUC for PE prediction at early gestation. CONCLUSION: Measurement of cfRNA can be used to effectively predict PE with high performance, providing an additional method for monitoring PE throughout the course of pregnancy.

Identification of Plasma hsa_circ_0001230 and hsa_circ_0023879 as Potential Novel Biomarkers for Focal Segmental Glomerulosclerosis and circRNA-miRNA-mRNA Network Analysis.

INTRODUCTION: Focal segmental glomerulosclerosis (FSGS) is a common glomerulopathy with an unclear mechanism. The demand for FSGS clinical diagnostic biomarkers has not yet been met. Circular RNA (circRNA) is a novel non-coding RNA with multiple functions, but its diagnostic value for FSGS remains unexplored. This study aimed to identify circRNAs that could aid in early clinical diagnosis and to investigate their mechanisms in podocyte injury. METHODS: The signature of plasma circRNAs for FSGS was identified by circRNA microarray. The existence of circRNAs was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR), RNase R assay, and DNA sequencing. Plasma levels of circRNAs were evaluated by qRT-PCR. The diagnostic value was appraised by the receiver operating characteristic curve. The circRNA-miRNA-mRNA network was built with Cytoscape 7.3.2. Statistically significant differences were calculated by the Mann-Whitney U test. RESULTS: A total of 493 circRNAs (165 upregulated, 328 downregulated) were differentially expressed in the plasma of FSGS patients (n = 3) and normal controls (n = 3). Eight candidate circRNAs were demonstrated to be circular and stable transcripts. Among them, hsa_circ_0001230 and hsa_circ_0023879 were significantly upregulated in FSGS patients (n = 29) compared to normal controls (n = 51). The areas under the curve value of hsa_circ_0001230 and hsa_circ_0023879 were 0.668 and 0.753, respectively, while that of the two-circRNA panel was 0.763. The RNA pull-down analysis revealed that hsa_circ_0001230 and hsa_circ_0023879 could sponge hsa-miR-106a. Additionally, hsa_circ_0001230 and hsa_circ_0023879 positively regulated hsa-miR-106a target genes phosphatase and tensin homolog (PTEN) and Bcl-2-like protein 11 (BCL2L11) in podocytes. CONCLUSION: hsa_circ_0001230 and hsa_circ_0023879 are novel blood biomarkers for FSGS. They may regulate podocyte apoptosis by competitively binding to hsa-miR-106a.

Development of a multigenomic liquid biopsy (PROSTest) for prostate cancer in whole blood.

INTRODUCTION: We describe the development of a molecular assay from publicly available tumor tissue mRNA databases using machine learning and present preliminary evidence of functionality as a diagnostic and monitoring tool for prostate cancer (PCa) in whole blood. MATERIALS AND METHODS: We assessed 1055 PCas (public microarray data sets) to identify putative mRNA biomarkers. Specificity was confirmed against 32 different solid and hematological cancers from The Cancer Genome Atlas (n = 10,990). This defined a 27-gene panel which was validated by qPCR in 50 histologically confirmed PCa surgical specimens and matched blood. An ensemble classifier (Random Forest, Support Vector Machines, XGBoost) was trained in age-matched PCas (n = 294), and in 72 controls and 64 BPH. Classifier performance was validated in two independent sets (n = 263 PCas; n = 99 controls). We assessed the panel as a postoperative disease monitor in a radical prostatectomy cohort (RPC: n = 47). RESULTS: A PCa-specific 27-gene panel was identified. Matched blood and tumor gene expression levels were concordant (r = 0.72, p < 0.0001). The ensemble classifier ("PROSTest") was scaled 0%-100% and the industry-standard operating point of ≥50% used to define a PCa. Using this, the PROSTest exhibited an 85% sensitivity and 95% specificity for PCa versus controls. In two independent sets, the metrics were 92%-95% sensitivity and 100% specificity. In the RPCs (n = 47), PROSTest scores decreased from 72% ± 7% to 33% ± 16% (p < 0.0001, Mann-Whitney test). PROSTest was 26% ± 8% in 37 with normal postoperative PSA levels (<0.1 ng/mL). In 10 with elevated postoperative PSA, PROSTest was 60% ± 4%. CONCLUSION: A 27-gene whole blood signature for PCa is concordant with tissue mRNA levels. Measuring blood expression provides a minimally invasive genomic tool that may facilitate prostate cancer management.