A metabolic map and artificial intelligence-aided identification of nasopharyngeal carcinoma via a single-cell Raman platform.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a complex cancer influenced by various factors. This study explores the use of single-cell Raman spectroscopy as a potential diagnostic tool for investigating biomolecular changes associated with NPC carcinogenesis. METHODS: Seven NPC cell lines, one immortalised nasopharyngeal epithelial cell line, six nasopharyngeal mucosa tissues and seven NPC tissue samples were analysed by performing confocal Raman spectroscopic measurements and imaging. The single-cell Raman spectral dataset was used to quantify relevant biomolecules and build machine learning classification models. Metabolomic profiles were investigated using ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). RESULTS: By generating a metabolic map of seven NPC cell lines, we identified an interplay of altered metabolic processes involving nucleic acids, amino acids, lipids and sugars. The results from spatially resolved Raman maps and UPLC-MS/MS metabolomics were consistent, revealing an increase of unsaturated fatty acids in cancer cells, particularly in highly metastatic 5-8F and poorly differentiated CNE2 cells. The classification model achieved a nearly perfect classification when identifying NPC and non-NPC cells with an ROC-AUC of 0.99 and a value of 0.97 when identifying 13 tissue samples. CONCLUSION: This study unveils a complex interplay of metabolic network and highlights the potential roles of unsaturated fatty acids in NPC progression and metastasis. This renders further research to provide deeper insights into NPC pathogenesis, identify new metabolic targets and improve the efficacy of targeted therapies in NPC. Artificial intelligence-aided analysis of single-cell Raman spectra has achieved high accuracies in the classification of both cancer cells and patient tissues, paving the way for a simple, less invasive and accurate diagnostic test.

Improving regional medical laboratory center report quality through a report recall management system.

OBJECTIVES: Currently, most medical laboratories do not have a dedicated software for managing report recalls, and relying on traditional manual methods or laboratory information system (LIS) to record recall data is no longer sufficient to meet the quality management requirements in the large regional laboratory center. The purpose of this article was to describe the research process and preliminary evaluation results of integrating the Medical Laboratory Electronic Record System (electronic record system) laboratory report recall function into the iLab intelligent management system for quality indicators (iLab system), and to introduce the workflow and methods of laboratory report recall management in our laboratory. METHODS: This study employed cluster analysis to extract commonly used recall reasons from laboratory report recall records in the electronic record system. The identified recall reasons were validated for their applicability through a survey questionnaire and then incorporated into the LIS for selecting recall reasons during report recall. The statistical functionality of the iLab system was utilized to investigate the proportion of reports using the selected recall reasons among the total number of reports, and to perform visual analysis of the recall data. Additionally, we employed P-Chart to establish quality targets and developed a "continuous improvement process" electronic flow form. RESULTS: The reasons for the recall of laboratory reports recorded in the electronic recording system were analyzed. After considering the opinions of medical laboratory personnel, a total of 12 recall reasons were identified, covering 73.05 % (1854/2538) of the recalled laboratory reports. After removing data of mass spectra lab with significant anomalies, the coverage rate increased to 82.66 % (1849/2237). The iLab system can generate six types of statistical graphs based on user needs, including statistical time, specialty labs (or divisions), test items, reviewers, reasons for report recalls, and distribution of the recall frequency of 0-24 h reports. The control upper limit of the recall rate of P-Chart based on laboratory reports can provide quality targets suitable for each professional group at the current stage. Setting the five stages of continuous process improvement reasonably and rigorously can effectively achieve the goal of quality enhancement. CONCLUSIONS: The enhanced iLab system enhances the intelligence and sustainable improvement capability of the recall management of laboratory reports, thus improving the efficiency of the recall management process and reducing the workload of laboratory personnel.

dbCNV: deleteriousness-based model to predict pathogenicity of copy number variations.

BACKGROUND: Copy number variation (CNV) is a type of structural variation, which is a gain or loss event with abnormal changes in copy number. Methods to predict the pathogenicity of CNVs are required to realize the relationship between these variants and clinical phenotypes. ClassifyCNV, X-CNV, StrVCTVRE, etc. have been trained to predict the pathogenicity of CNVs, but few studies have been reported based on the deleterious significance of features. RESULTS: From single nucleotide polymorphism (SNP), gene and region dimensions, we collected 79 informative features that quantitatively describe the characteristics of CNV, such as CNV length, the number of protein genes, the number of three prime untranslated region. Then, according to the deleterious significance, we formulated quantitative methods for features, which fall into two categories: the first is variable type, including maximum, minimum and mean; the second is attribute type, which is measured by numerical sum. We used Gradient Boosted Trees (GBT) algorithm to construct dbCNV, which can be used to predict pathogenicity for five-tier classification and binary classification of CNVs. We demonstrated that the distribution of most feature values was consistent with the deleterious significance. The five-tier classification model accuracy for 0.85 and 0.79 in loss and gain CNVs, which proved that it has high discrimination power in predicting the pathogenicity of five-tier classification CNVs. The binary model achieved area under curve (AUC) values of 0.96 and 0.81 in the validation set, respectively, in gain and loss CNVs. CONCLUSION: The performance of the dbCNV suggest that functional deleteriousness-based model of CNV is a promising approach to support the classification prediction and to further understand the pathogenic mechanism.

The Comparison of Biomolecular Changes of Epstein-Barr Virus Infection in Nasopharyngeal Epithelial Cells Using Confocal Raman Microspectroscopy.

BACKGROUND: Due to its unique fingerprinting properties, Confocal Raman microscopy (CRM) can be used to examine the biomolecular changes of viruses invading and manipulating host cells. Recently, the biochemical changes due to the invasion and infection of B lymphocyte cells, nerve cells, and epithelial cells by Epstein-Barr virus (EBV) have been reported. However, biomolecular changes in nasopharyngeal epithelial cells that result from EBV infection are still poorly understood. METHODS: In continuation of our prior investigation of EBV infection in nasopharyngeal epithelial cells, we tried to expound on biomolecular changes in EBV-infected nasopharyngeal epithelial cells using Raman microspectroscopy. EBV has two life cycles, latent infection and lytic replication. We have established latent and lytic infection models at the cellular level. In order to understand the characteristics of the two patterns of EBV infection, we used Raman spectroscopy to identify the changes in biomolecules of EBV latent cells (CNE2, CNE2-EBV) and lytic cells (NPEC1-BMI1-CN, NPEC1-BMI1-EBV). RESULTS: During latent infection, levels of glycogen, protein, and lipid molecules in the cell increased while levels of nucleic acid and collagen molecules decreased. Molecular levels of glycogen, proteins, and nucleic acids are reduced during lytic infection. We found that molecular levels of nucleic acid decreased during two different periods of infection, whereas levels of other biomolecules showed the opposite trend. Glycogen, proteins, lipids, nucleic acids, and other molecules are associated with alterations in cellular biochemical homeostasis. These changes correspond to unique Raman spectra in infected and uninfected cells associated with specific biomolecules that have been proven. These molecules are mainly responsible for cellular processes such as cell proliferation and apoptosis. The Raman signatures of these biomolecular changes depend on the different phases of viral infection. CONCLUSIONS: Therefore, by using CRM, it is possible to discern details in the progression of EBV infection in nasopharyngeal epithelial cells at the molecular level.

Comprehensive analysis of aberrantly expressed circRNAs, mRNAs and lncRNAs in patients with nasopharyngeal carcinoma.

BACKGROUND: The location of nasopharyngeal cancer is hidden, so it is difficult to diagnose at an early stage. In this study, we aimed to investigate the expression profiles of circRNAs, mRNAs and IncRNAs and to provide some basis for further studies. METHODS: Expression profiles of circRNAs, mRNAs, and lncRNAs were analyzed using microarray techniques. The differentially expressed ncRNA was calculated by bioinformatics. RESULTS: A total of 3048 circRNAs, 2179 lncRNAs, and 2015 mRNAs were detected to be significantly differentially expressed in NPC. The most upregulated circRNAs, lncRNAs, and mRNAs were hsa-circ-0067562, NONHSAT232922.1, and HOXB13, respectively. And, the most downregulated circRNAs, lncRNAs, and mRNAs were hsa_circ_0078837, lnc-TTC8-4:3, and LTF, respectively. The number of upregulated DE lncRNAs was more than twice than those downregulated. Our data showed that 80.44% of pairs of lncRNAs and cis-mRNAs demonstrated positive correlations. For lncRNAs and trans-mRNAs pairs, 53.7% of pairs showed positive correlation. LncRNA-mediated cis regulation is a prevalent regulatory mode in the development of nasopharyngeal carcinoma. CR1, LRMP and SORBS2 are predicted to be mediated not only by cis-acting lncRNA modes of action, but also by trans-acting lncRNA mechanisms. Additionally, we constructed a diagnostic prediction model with a high sensitivity and specificity. CONCLUSION: Our study characterized the landscape of circRNAs, mRNAs and lncRNAs in NPC tissue and provided novel insights into the molecular mechanisms of NPC.

Segmental aneuploidies with 1 Mb resolution in human preimplantation blastocysts.

PURPOSE: This study aimed to investigate the spectrum and characteristics of segmental aneuploidies (SAs) of <10 megabase (Mb) length in human preimplantation blastocysts. METHODS: Preimplantation genetic testing for aneuploidy was performed in 15,411 blastocysts from 5171 patients using a validated 1 Mb resolution platform. The characteristics and spectrum of SAs, including the incidence, sizes, type, inheritance pattern, clinical significance, and embryo distribution, were studied. RESULTS: In total, 6.4% of the 15,411 blastocysts carried SAs of >10 Mb, 4.9% of embryos had SAs ranging between 1 to 10 Mb, and 84.3% of 1 to 10 Mb SAs were <5 Mb in size. Inheritance pattern analysis indicated that approximately 63.8% of 1 to 10 Mb SAs were inherited and were predominantly 1 to 3 Mb in size. Furthermore, 18.4% of inherited SAs and 51.9% de novo 1 to 10 Mb SAs were pathogenic or likely pathogenic (P/LP). Different from whole-chromosome aneuploidies, reanalysis indicated that 50% of the de novo 1 to 10 Mb SAs and 70% of the >10 Mb SAs arose from mitotic errors. CONCLUSION: Based on the established platform, 1 to 10 Mb SAs are common in blastocysts and include a subset of P/LP SAs. Inheritance pattern analysis and clinical interpretation based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines contributed to determine the P/LP SAs.

Analytical performance evaluation of five RT-PCR kits for severe acute respiratory syndrome coronavirus 2.

BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.

Consistency evaluation of Liferiver, Yaneng, Darui, and the Cobas 4800 test for high-risk human papillomavirus screening.

BACKGROUND: In recent years, several high-risk human papillomavirus (HR-HPV) tests have been developed. The assay capabilities need to be systematically reviewed. Here, we compared the clinical sample performance of three novel HR-HPV assays (Liferiver, Yaneng, and Darui) based on different platforms with the widely adopted cobas4800 test. METHODS: A total of 346 cervical swabs from women who were screened for cervical cancer were analyzed for the presence of 14 HR-HPV genotypes. The distinction between the four assays was investigated by the genotyping and direct sequencing. RESULTS: The positive rates of the four assays ranged from 61.56% to 64.16%. The overall concordance was 88.15%. The Yaneng assays displayed the best sensitivity (100%) and specificity (98.43%). The sensitivity (98.17%) and specificity (98.43%) of the Darui assay were superior to those of the cobas4800 test (97.72% and 93.70%, respectively). The Liferiver assay displayed comparable sensitivity with the cobas4800 test (95.89% and 97.72%, respectively). The specificity of the cobas4800 was lower than that of the Liferiver assay (93.70% vs. 97.64%). CONCLUSIONS: The three novel HR-HPV assays displayed good agreement with the cobas4800 test. The analytical performance of all four fulfilled the requirements of sensitivity and specificity for HR-HPV detection.

Profiling of downregulated blood-circulating miR-150-5p as a novel tumor marker for cholangiocarcinoma.

Altered microRNA (miRNA) expression plays a role in cholangiocarcinoma (CCA) development; thus, detection of blood-circulating miRNAs could be useful as CCA markers. This study profiled serum miRNA levels in patients with primary sclerosing cholangitis (PSC) and CCA and then assessed the role of miR-150-5p in CCA progression in vitro. Three samples were randomly selected from each of 50 sera of healthy controls, 30 PSC sera, and 28 CCA sera with matched bile samples for miRNA microarray profiling. The dysregulated miRNAs were confirmed using qRT-PCR, and miR-150-5p was selected for further in vitro and ex vivo studies. The miRNA microarray identified three dysregulated miRNAs in both CCA and PSC samples, while miR-150-5p level was consistently lower in CCA sera, bile, and tissues than in normal control and PSC sera (P < 0.05). Furthermore, levels of miR-150-5p were associated with serum carbohydrate antigen 19-9 (CA19-9) levels and CCA pathological grade. Bioinformatic Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses showed that miR-150-5p could regulate hand-full gene pathways, including cancer pathway (P < 0.01). However, overexpression of miR-150-5p inhibited proliferation, migration, and invasion capability of CCA cells (P < 0.05). Luciferase reporter assay showed that miR-150-5p bound to an oncogene Ets including gene-1 (ELK1), and Western blot data confirmed that miR-150-5p suppressed ELK1 expression in CCA cell lines. These results suggest that reduced miR-150-5p expression could contribute to CCA development and progression due to uncontrolled ELK1 expression. Thus, further study could evaluate miR-150-5p as a novel target and predictor for CCA prevention and treatment.

Co-expression of COX-2 and 5-LO in primary glioblastoma is associated with poor prognosis.

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) are important factors in tumorigenesis and malignant progression; however, studies of their roles in glioblastoma have produced conflicting results. To define the frequencies of COX-2 and 5-LO expression and their correlation with clinicopathological features and prognosis, tumor tissues from 76 cases of newly diagnosed primary ordinary glioblastoma were examined for COX-2 and 5-LO expression by immunohistochemistry. The expression levels of COX-2 and 5-LO and the relationships between the co-expression of COX-2/5-LO and patient age and gender, edema index (EI), Karnofsky Performance Scale and overall survival (OS) were analyzed. COX-2 and 5-LO were expressed in 73.7 % (56/76) and 92.1 % (70/76) of the samples, respectively. Among the clinicopathological characteristics, only age (>60 years) exhibited a significant association with the high expression of COX-2. No statistically significant correlations were found in the 5-LO cohort. A significant positive correlation was revealed between the COX-2 and 5-LO scores (r = 0.374; p = 0.001). The elevated co-expression of COX-2 and 5-LO was observed primarily in the patients over the age of 60 years. Patients with a high expression of COX-2 had a significantly shorter OS (p < 0.01), whereas the immunoexpression of 5-LO was not associated with the OS of patients with glioblastoma. Survival analysis indicated that simultaneous high levels of COX-2 and 5-LO expression were significantly correlated with poor OS and, conversely, that a low/low expression pattern of these two proteins was significantly associated with better OS (p < 0.05). Moreover, the Cox multivariable proportional hazard model showed that a high expression of COX-2, high co-expression of COX-2 and 5-LO, and a high Ki-67 index were significant predictors of shorter OS in primary glioblastoma, independent of age, gender, EI, 5-LO expression and p53 status. The hazard ratios for OS were 2.347 (95 % CI 1.30-4.25, p = 0.005), 1.900 (95 % CI 1.30-2.78, p = 0.001), and 2.210 (95 % CI 1.19-4.09, p = 0.011), respectively. These results suggest that COX-2 and 5-LO play roles in tumorigenesis and the progression of primary glioblastoma and that the co-expression pattern of COX-2/5-LO may be used as an independent prognostic factor in this disease.

Early Biomarkers and Hearing Impairments in Patients with Neonatal Hypoxic-Ischemic Encephalopathy.

Identifying biomarkers for hearing impairments (HIs) in patients with neonatal hypoxic-ischemic encephalopathy (HIE), to initialize early hearing habilitation, is crucial. Seventy-eight neonates with HIE were divided into the following two groups: those with HIs and those without HIs. We compared those patients with 11,837 newborns without HIE, and analyzed the risk factors of HIs among neonatal HIE. Of the 78 patients, 11 were confirmed to have an HI, which is a substantially higher percentage than in the 11,837 newborns without HIE (14.1% vs. 0.87%; p < 0.001). More patients with moderate-to-severe HIE had confirmed HIs (p = 0.020; odds ratio, 8.61) than those with mild HIE. Clinical staging, and blood lactate and glucose levels could be predictive factors for HIs among patients with HIE. The patients who exhibited HIs had significantly higher lactate (104.8 ± 51.0 vs. 71.4 ± 48.4; U = 181, p = 0.032) and serum glucose (159.5 ± 86.1 vs. 112.1 ± 62.3; U = 166, p = 0.036) levels than those without HIs. A higher prevalence of HIs was noted in the patients with stage III HIE than those with stage II HIE (43.8% vs. 10%; p = 0.008). The degree of HI correlated with brain anomalies and neurodevelopmental outcomes at 1 year of age. Clinical staging, and blood lactate and glucose levels could be predictive factors for HIs among patients with HIE.

The Overexpression of NMHC IIA Promoted Invasion and Metastasis of Nasopharyngeal Carcinoma Cells.

Background: Nasopharyngeal carcinoma (NPC) is a kind of head and neck squamous cell carcinoma (HNSCC) with a strong tendency for metastasis and recurrence. Non-muscle myosin heavy chain IIA (NMHC IIA) plays important roles in recurrence and metastasis of cancers. However, the function and mechanism of NMHC IIA expression in NPC remain unclear. Methods: A receiver operating characteristic (ROC) curve was constructed for 141 specimens of HNSCC tissues and 44 control samples from The Cancer Genome Atlas (TCGA) database. Co-expressed genes with MYH9 were identified using LinkedOmics. Transcription factors (TFs) and miRNA regulation network were constructed using Networkanalyst. The migration and invasion ability of nasopharyngeal carcinoma cells were evaluated by in vitro migration and matrigel invasion assays, respectively. Results: The public microarray results showed that MYH9 expression levels were upregulated in HNSCC tissues compared with the matched adjacent normal tissues in this study (p<0.0001). The AUC of MYH9 reached up to 0.8303 at a cutoff value of 175.2, with a sensitivity and specificity of 70.21% and 86.36%, respectively. MYH9 expression was increased in lymph node metastasis HNSCC tumors compared with that in tumors without lymph node metastasis (p<0.05) and showed a strong positive association with expression of FLNA. High MYH9 and FLNA expression were related with poorer overall survival in HNSCC. MYH9 with positively associated genes regulated focal adhesion, cell-substrate junction assembly and cell morphogenesis were involved in differentiation using GO and KEGG analysis. MYH9 was correlated with a network of TFs including SP1, SRF, JUN and FOS in HNSCC. The suppression of endogenous NMHC IIA decreased cellular migration and invasion in HNE1 cells and reduced the expression of phosphorylation of EGFR, AKT and ERK. The over-expression of NMHC IIA increased cellular migration and invasion in COS-7 cells and increased the expression of phosphorylation of EGFR, AKT and ERK. Conclusion: Expression of NMHC IIA mRNA was higher in HNSCC than in the adjacent normal tissues. NMHC IIA expression was increased in lymph node metastasis HNSCC tumors compared with tumors without lymph node metastasis. High MYH9 was association with poorer overall survival in HNSCC. NMHC IIA expression increased the invasion and metastasis abilities of the nasopharyngeal cancer cell line in vitro by augmenting the expression of phosphorylation of EGFR, AKT and ERK. These findings will be beneficial for providing an effectively therapeutic strategy for NPC.

A Novel Diagnostic Biomarker, PZP, for Detecting Colorectal Cancer in Type 2 Diabetes Mellitus Patients Identified by Serum-Based Mass Spectrometry.

Background: Growing evidence has confirmed that populations with type 2 diabetes mellitus (T2DM) have an increasing risk of developing colorectal cancer (CRC). Thus, convenient and effective screening strategies for CRC should be developed for the T2DM population to increase the detection rate of CRC. Methods: Twenty serum samples extracted from five healthy participants, five T2DM patients, five CRC patients and five T2DM patients with CRC (T2DM + CRC) were submitted to data-independent acquisition mass spectrometry (DIA-MS) analysis to discover unique differentially altered proteins (DAPs) for CRC in patients with T2DM. Then, the diagnostic value of pregnancy zone protein (PZP) was validated by ELISA analysis in the validated cohort. Results: Based on DIA-MS analysis, we found eight unique proteins specific to T2DM patients with CRC. Among these proteins, four proteins showed different expression between the T2DM + CRC and T2DM groups, and PZP exhibited the largest difference. Next, the diagnostic value of serum PZP was validated by ELISA analysis with an AUC of 0.713. Moreover, the combination of PZP, CA199 and CEA exhibited encouraging diagnostic value, and the AUC reached 0.916. Conclusion: Overall, our current research implied that PZP could be regarded as a newfound serum biomarker for CRC medical diagnosis in T2DM patients.

miR-224 Regulates the Aggressiveness of Hepatoma Cells Through the IL-6/STAT3/SMAD4 Pathway.

BACKGROUND: Previous studies have shown that miR-224 regulates the progression of liver cancer. The aim of this study was to investigate the underlying mechanisms. METHODS: The miR-224, p-STAT3 and SMAD4 expression levels were checked with tissue or/and serum samples of HCC patients by qRT-PCR or IHC methods. The regulatory role of IL-6 in p-STAT3 and SMAD4 was investigated by Western-blot. The targeted gene of miR-224 was verified by both Western-blot and luciferase reporter assay. Furthermore, the carcinogenesis of miR-224 in HCC was investigated by cell experiments in vitro and mouse xenograft model and in vivo imaging in vivo. RESULTS: It was found miR-224 was elevated in both tissue and serum of HCC patients. The p-STAT3 expression was higher but the SMAD4 was lower in the HCC tumor tissues. Moreover, IL-6 can induce the p-STAT3/STAT3 and miR-224 expression in HCC cells and STAT3 played the bridge role between IL-6 and miR-224. Target gene studies found miR-224 targeted the 3'UTR of SMAD4. Finally, the promoting roles of miR-224in the growth, proliferation, invasion and migration of HCC were discovered by in vitro and in vivo studies. CONCLUSION: It implies that miR-224 may potentially represent a new target for developing novel anti-HCC therapeutics.

An aberrant DNA methylation signature for predicting the prognosis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy worldwide with a poor prognosis. DNA methylation is an epigenetic modification that plays a critical role in the etiology and pathogenesis of HNSCC. The current study aimed to develop a predictive methylation signature based on bioinformatics analysis to improve the prognosis and optimize therapeutic outcome in HNSCC. Clinical information and methylation sequencing data of patients with HNSCC were downloaded from The Cancer Genome Atlas database. The R package was used to identify differentially methylated genes (DMGs) between HNSCC and adjacent normal tissues. We identified 22 DMGs associated with 246 differentially methylated sites. Patients with HNSCC were classified into training and test groups. Cox regression analysis was used to build a risk score formula based on the five methylation sites (cg26428455, cg13754259, cg17421709, cg19229344, and cg11668749) in the training group. The Kaplan-Meier survival curves showed that the overall survival (OS) rates were significantly different between the high- and low-risk groups sorted by the signature in the training group (median: 1.38 vs. 1.57 years, log-rank test, p < 0.001). The predictive power was then validated in the test group (median: 1.34 vs. 1.75 years, log-rank test, p < 0.001). The area under the receiver operating characteristic curve (area under the curve) based on the signature for predicting the 5-year survival rates, was 0.7 in the training and 0.73 in test groups, respectively. The results of multivariate Cox regression analysis showed that the riskscore (RS) signature based on the five methylation sites was an independent prognostic tool for OS prediction in patients. In addition, a predictive nomogram model that incorporated the RS signature and patient clinical information was developed. The innovative methylation signature-based model developed in our study represents a robust prognostic tool for guiding clinical therapy and predicting the OS in patients with HNSCC.

MicroRNA-23b-3p promotes pancreatic cancer cell tumorigenesis and metastasis via the JAK/PI3K and Akt/NF-κB signaling pathways.

MicroRNA (miR)-23b-3p plays an important role in tumor growth, proliferation, invasion and migration in pancreatic cancer (PC). However, the function and mechanistic role of miR-23b-3p in the development of PC remains largely unknown. In the present study, the miR-23b-3p levels in the serum of patients with PC were found to be elevated, and the phosphorylation levels of Janus kinase (JAK)2, PI3K, Akt and NF-κВ were found to be upregulated. In addition, miR-23b-3p was induced in response to interleukin-6 (IL-6), which is known to be involved in the progression of PC. Overexpression of miR-23b-3p, on the other hand, activated the JAK/PI3K and Akt/NF-κB signaling pathways in PC cells, as evidenced by miR-23b-3p-induced upregulation of phosphorylated (p-)JAK2, p-PI3K, p-Akt and p-NF-κВ, as well as the downregulation of PTEN; and these effects were found to be reversible by miR-23b-3p inhibition. Furthermore, miR-23b-3p was found to downregulate PTEN by directly targeting the 3'-untranslated region of PTEN mRNA. Notably, in an in vivo xenograft mouse model, overexpression of miR-23b-3p accelerated PC cell-derived tumor growth, activated the JAK/Akt/NF-κВ signaling pathway and promoted liver metastasis. In contrast, knockdown of miR-23b-3p suppressed tumor growth and metastasis as well as JAK/Akt/NF-κВ signaling activity. In vivo imaging of the mice further confirmed the metastasis promoting role of miR-23b-3p in PC. These results suggested that miR-23b-3p enhances PC cell tumorigenesis and metastasis, at least, partially via the JAK/PI3K and Akt/NF-κB signaling pathways. Therefore, targeting miR-23b-3p or the JAK/PI3K and Akt/NF-κB signalings may be potential therapeutic strategy against PC.

LINC00958 and HOXC13-AS as key candidate biomarkers in head and neck squamous cell carcinoma by integrated bioinformatics analysis.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor with a strong tendency for metastasis and recurrence. Finding effective biomarkers for the early diagnosis of HNSCC is critical for the early treatment and prognosis of patients. METHODS: RNA sequencing data including long non-coding RNAs (lncRNAs), messenger RNA (mRNAs) and microRNAs (miRNAs) of 141 HNSCC and 44 adjacent normal tissues were obtained from the TCGA. Differentially expressed genes were analyzed using the R package DESeq. GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. A competing endogenous RNAs (ceRNA) network was constructed. The most differentially expressed genes in the main ceRNA network were chosen for nasopharyngeal carcinoma (NPC) cell lines and NPEC2 Bmi-1 cell line verification. A receiver operating characteristic (ROC) curve was constructed for 141 specimens of HNSCC tissues from 44 control samples. RESULTS: In our study, 79 HNSCC-associated abnormally expressed lncRNAs , 86 abnormally expressed miRNAs and 324 abnormally expressed mRNAs were identified. The public microarray results showed that LINC00958 and HOXC13-AS expression levels were upregulated in HNSCC tissues compared with the adjacent normal tissues in this study (p < 0.0001). LINC00958 and HOXC13-AS expression levels in NPC cell lines were higher than those in the NPEC2 Bmi-1 cell line (p < 0.05). The results showed that the area under the ROC curve (AUC) of LINC00958 reached up to 0.906 at a cutoff value of 7.96, with a sensitivity and specificity of 80.85% and 90.91%, respectively. The AUC of HOXC13-AS reached up to 0.898 at a cutoff value of 0.695, with sensitivity and specificity values of 86.23% and 83.78%, respectively. CONCLUSION: The current study indicates that LINC00958 and HOXC13-AS are new candidate diagnostic biomarkers for HNSCC patients.

Development of a novel prognostic signature for predicting the overall survival of bladder cancer patients.

BACKGROUND: Bladder cancer is one of the most common malignancies. So far, no effective biomarker for bladder cancer prognosis has been identified. Aberrant DNA methylation is frequently observed in the bladder cancer and holds considerable promise as a biomarker for predicting the overall survival (OS) of patients. MATERIALS AND METHODS: We downloaded the DNA methylation and transcriptome data for bladder cancer from The Cancer Genome Atlas (TCGA), a public database, screened hypo-methylated and up-regulated genes, similarly, hyper-methylation with low expression genes, then retrieved the relevant methylation sites. Cox regression analysis was used to identify a nine-methylation site signature of a training group. Predictive ability was validated in a test group by receiver operating characteristic (ROC) analysis. RESULTS: We identified nine bladder cancer-specific methylation sites as potential prognostic biomarkers and established a risk score system based on the methylation site signature to evaluate the OS. The performance of the signature was accurate, with area under curve was 0.73 in the training group and 0.71 in the test group. Taking clinical features into consideration, we constructed a nomogram consisting of the nine-methylation site signature and patients' clinical variables, and found that the signature was an independent risk factor. CONCLUSIONS: Overall, the significant nine methylation sites could be novel prediction biomarkers, which could aid in treatment and also predict the overall survival likelihoods of bladder cancer patients.

Up-regulation of miR-155 contributes to TNF-mediated hepatocyte apoptosis in acute liver failure.

BACKGROUND/AIMS: Acute liver failure (ALF) is due to severe immune response, resulting in massive apoptosis/necrosis of hepatocytes. The precise mechanism has not been explored yet. MATERIALS AND METHODS: The mouse with ALF model was induced by D-GalN/LPS; the hepatic miRNAs expression profile was evaluated by miRNA microarray and verified by RT-PCR. During the ALF in mice, the miR-155 expression was detected in the liver as well as in spleen. Then the correlation between miR-155 and inflammatory cytokines was evaluated. Furthermore, the miR-155 expression in activated Raw264.7 cells and apoptotic hepatocytes was also studied. Finally, the regulatory roles of miR-155 in TNF expression of apoptotic hepatocytes were shown. RESULTS: It was shown that miRNAs changed in the mice with ALF relating to hepatocytes apoptosis/necrosis; the selected miRNAs were confirmed with RT-PCR. miR-155 was up-regulated, but miR-698, -720, and -329 were down-regulated. Moreover, hepatic miR-155 was up-regulated at all-time points in the liver, but only at 7 h in spleen of mice with ALF. A significant correlation was observed between hepatic miR-155 and TNF/IL-6 in mice with ALF, which was supported by the findings in vitro showing up-regulated miR-155 in Raw264.7 cells and Hepa1-6 cells under LPS or D-GalN+TNF induction, respectively. Moreover, a correlation was observed between miR155 and TNF levels in vivo and in vitro. CONCLUSION: These data demonstrate that miR-155 regulates TNF-mediated hepatocyte apoptosis in ALF, which provides some useful information in both basic and clinical researches.