Combined metabolomic and lipidomic analysis uncovers metabolic profile and biomarkers for papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy with a rapidly increasing incidence. The pathogenesis of PTC is unclear, but metabolic and lipidomic reprogramming may play a role in tumor growth. We applied ultra-performance liquid chromatography-tandem mass spectrometry to perform widely targeted metabolomics and lipidomics on plasma samples from 94 patients with PTC and 100 healthy controls. We identified 113 differential metabolites and 236 differential lipids, mainly involved in branched-chain amino acid metabolism, glutamate and glutamine metabolism, tricarboxylic acid cycle, and lipid metabolism. We also screened three potential metabolite biomarkers: sebacic acid, L-glutamine, and indole-3-carboxaldehyde. These biomarkers showed excellent diagnostic performance for PTC in both discovery and validation cohorts, with areas under the receiver operating characteristic curves of 0.994 and 0.925, respectively. Our findings reveal distinct metabolic and lipidomic features of PTC and provide novel targets for diagnosis and treatment.

IGF2BP2-induced circRUNX1 facilitates the growth and metastasis of esophageal squamous cell carcinoma through miR-449b-5p/FOXP3 axis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive malignancies with relatively high morbidity and mortality. Emerging evidence suggests circular RNAs (circRNAs) play critical roles in tumor cell malignancy. However, the biological function and clinical significance of many circRNAs in ESCC remain elusive. METHODS: The expression level and clinical implication of circRUNX1 in ESCC tissues were evaluated using qRT-PCR. In vitro and in vivo functional studies were conducted to investigate the underlying biological effects of circRUNX1 on ESCC cell growth and metastasis. Moreover, bioinformatics analysis, RNA sequencing (RNA-seq), RNA immunoprecipitation (RIP) assays, dual-luciferase reporter assays, and rescue experiments were performed to explore the relationships between circRUNX1, miR-449b-5p, Forkhead box protein P3 (FOXP3), and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RESULTS: CircRUNX1 was found to be significantly up-regulated in ESCC tissues and associated with TNM stage and differentiation grade. Functionally, circRUNX1 promoted ESCC cell proliferation and metastasis in vitro and in vivo. CircRUNX1 enhanced FOXP3 expression by competitively sponging miR-449b-5p. Notably, both miR-449b-5p mimics and FOXP3 knockdown restored the effects of circRUNX1 overexpression on cell proliferation and metastasis. Furthermore, IGF2BP2 binding to circRUNX1 prevented its degradation. CONCLUSIONS: IGF2BP2 mediated circRUNX1 functions as an oncogenic factor to facilitate ESCC progression through the miR-449b-5p/FOXP3 axis, implying that circRUNX1 has the potential to be a promising diagnostic marker and therapeutic target for ESCC patients.

LncRNA LOXL1-AS1 promotes esophageal squamous cell carcinoma progression by targeting DESC1.

Recently, ample evidence indicated that numerous aberrantly expressed long non-coding RNAs (lncRNAs) participated in the development of multiple malignancies. However, the expression and function of lncRNA LOXL1-AS1 in mediating esophageal squamous cell carcinoma (ESCC) carcinogenesis remains largely elusive. Here we validated that LOXL1-AS1 was significantly upregulated in ESCC tissues compared with the corresponding adjacent non-neoplastic tissues, and LOXL1-AS1 expression was positively correlated with ESCC patients' lymph node metastasis. Besides, LOXL1-AS1 knockdown impaired ESCC cells proliferation, migration and invasion capabilities in vitro. Furthermore, inhibiting LOXL1-AS1 in ESCC cells increased the percentage of cells at the G1 phase, accompanied by reducing in S phase in contrast to scramble control, and silencing of LOXL1-AS1 evoked ESCC cell apoptosis. From high throughput RNA sequencing (RNA-seq) analysis, we identified that differentially expressed in squamous cell carcinoma 1 (DESC1) was a critical downstream target of LOXL1-AS1. Taken together, we demonstrated the function and mechanism of LOXL1-AS1 in contributing ESCC progression for the first time, and indicated LOXL1-AS1 may be a novel therapeutic biomarker of ESCC.

TMT proteomics analysis of intestinal tissue from patients of irritable bowel syndrome with diarrhea: Implications for multiple nutrient ingestion abnormality.

Diarrheal irritable bowel syndrome (IBS-D) is a chronic functional bowel disease with no clear diagnostic markers and no satisfactory treatment strategies. In recent years, the importance of intestinal microstructure and function in IBS-D has been emphasized. However, the intestinal tissue proteomics of IBS-D patients has not been analyzed. Here, we systematically analyzed the molecule profiling of the intestinal tissues in IBS-D patients through tandem mass tag (TMT)-based proteomics for the first time, aiming to reveal the pathogenesis and provide evidence for diagnosis and treatment of IBS-D. Five IBS-D patients and five healthy subjects were selected, biopsy tissue samples from the junction of sigmoid and rectum were analyzed by TMT proteomics. Differentially expressed proteins were obtained and bioinformatics analysis was performed. Furthermore, parallel reaction monitoring (PRM) and q-PCR detection were applied to validate the differentially expressed proteins. Eighty differentially expressed proteins were screened, 48 of which were up-regulated and 32 were down-regulated (fold change >1.2, P < 0.05). Bioinformatics analysis showed that these proteins were significantly enriched in the nutrient ingestion pathways which are related to immune molecules. SELENBP1, VSIG2, HMGB1, DHCR7, BCAP31 and other molecules were significantly changed. Our study revealed the underlying mechanisms of IBS-D intestinal dysfunction. SIGNIFICANCE: Irritable bowel syndrome with diarrhea (IBS-D) is a worldwide chronic intestinal disease with no definite diagnostic markers. It is still a challenge to accurately locate the pathogenesis of patients for appropriate treatment strategy. Established proteomics studies of IBS-D are only based on urine, blood, or tissue samples from animals. Our study was the first TMT proteomics analysis on intestinal biopsy tissues of patients with IBS-D, which revealed the changes of molecular spectrum of actual intestinal conditions in patients with IBS-D. Some important molecules and signaling pathways have been found abnormal in our study, which were related with nutrient uptake. They not only provided preliminary clues for low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) intolerance, an unsolved conundrum of IBS-D, but also revealed obscure problems of protein, lipid, and other nutrients ingestion in IBS-D patients. Some of these differentially expressed molecules have been preliminarily verified, and will may be potential candidate molecules for diagnostic markers of IBS-D.

From Genotype to Phenotype: Content and Activities of Cytochromes P450 2A6 in Human Liver In Vitro and Predicted In Vivo.

Unraveling the molecular mechanisms by which genetic variants of cytochrome P450 2A6 lead to different metabolic phenotypes remains a long-standing but important challenge. CYP2A6 is an enzyme involved in the metabolism of several clinical drugs as well as the metabolic activation of carcinogenic nitrosamines. Herein, CYP2A6 genotypes and phenotypes, as indicated by protein content [by liquid chromatography-mass spectrometry (MS)/MS] and metabolic activities [Vmax, clearance (CL)], were determined for 90 human liver samples. We determined the median, range, and interindividual and intraindividual variation of CYP2A6 content and activity at the microsomal, liver tissue, and whole liver level and predicted hepatic in vivo clearance by in vitro-in vivo extrapolation based on CYP2A6-mediated coumarin metabolism by each CYP2A6 genotype. These results reveal how different CYP2A6 genotypes yield different phenotypic traits in protein content and enzyme activity. For relative Vmax, CL, and protein content, the intraindividual percentage coefficients of variation (ICVs) were 41.0% (18.8%-125.1%), 28.5% (2.39%-133.5%), and 27.8% (2.68%-88.0%), respectively. The high ICVs implied large intraindividual variation at different levels, sometimes in a genotype-dependent manner. Intergenotype analysis revealed that the CYP2A6*4 allele demonstrated the most obvious effect on phenotypic outcomes, both in protein content and in metabolic activity. Indeed, decreased CYP2A6 protein content with the CYP2A6*4 genotype might explain the decreased metabolic activity from the molecular to the organismal level. These findings may allow useful predictions for CYP2A6-mediated drug metabolism on an individual patient basis in accord with the goal of achieving personalized medicine. SIGNIFICANCE STATEMENT: We provide the median, range, and interindividual and intraindividual variation in CYP2A6 content at the microsomal, liver tissue, and whole liver level by liquid chromatography-mass spectrometry (MS)/MS as well as activities at the protein, microsomal, liver tissue, and whole liver level both in vitro and at the organismal level based on CYP2A6-mediated coumarin metabolism with each CYP2A6 genotype, thereby allowing us to elucidate how different CYP2A6 genotypes yield differing phenotypic traits (protein content and enzyme activity), facilitating the development of personalized medicine.

LncRNA MNX1-AS1 promotes progression of esophageal squamous cell carcinoma by regulating miR-34a/SIRT1 axis.

BACKGROUND: Long non-coding RNAs (lncRNAs) are powerful factors influencing the tumorigenesis and metastasis of multiple carcinomas. LncRNA MNX1-AS1 plays critical roles in the progression of tumor formation according to recent research, while its roles in esophageal squamous cell carcinoma (ESCC) remains unknown. METHODS: The expression levels of lncRNA MNX1-AS1 were examined in ESCC tissues by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The role of lncRNA MNX1-AS1 was performed by WST-1 proliferation assays, migration and invasion assays. Besides, the molecular mechanism of lncRNA MNX1-AS1 was verified by online bioinformatics, qRT-PCR and rescue assays. RESULTS: MNX1-AS1 was signifcantly upregulated in ESCC tissues. It was conformed that high MNX1-AS1 expression was associated with ESCC lymph node metastasis. Moreover, we found that knockdown of MNX1-AS1 apparently suppressed the cell proliferation, migration, and invasion capacity. Flow cytometry analysis showed MNX1-AS1 regulated ESCC cell cycle and apoptosis progression. Mechanism analysis revealed that miR-34a inhibitor could rescue the influence of inhibiting MNX1-AS1 on ESCC cells migration by serving as competing endogenous RNA (ceRNAs). Furthermore, we found that miR-34a specifically targeted SIRTI. CONCLUSIONS: Taken together, we demonstrated that lncRNA MNX1-AS1/miR-34a/SIRT1 regulatory axis could play an important role in ESCC progression, and MNX1-AS1 may act as a novel potential biomarker for esophageal squamous cell carcinoma.

Intraindividual Variation and Correlation of Cytochrome P450 Activities in Human Liver Microsomes.

We systematically studied and explored intraindividual variation and correlation in the activities of cytochrome P450 (CYP) using 105 normal liver samples. The intraindividual percentage coefficients of variation of relative Km, Vmax, and CLint were 45.9% (18.3-140%), 44.0% (16.8-127%), and 52.0% (24.2-134%). Overall values of relative Km, Vmax, and CLint for 10 CYPs were sorted. The order, from highest to lowest, was CYP2D6, 3A4/5, 2C19, 2C9, 2B6, 1A2, 2A6, 2C8, and 2E1 for Km; CYP3A4/5, 2C19, 2D6, 2C8, 2E1, 2B6, 1A2, 2A6, and 2C9 for Vmax; and CYP2D6, 2B6, 3A4/5, 2C19, 2C9, 1A2, 2E1, 2C8, and 2A6 for CLint. CYP2A6*4, 2B6 785A>G, and 2D6 100C>T contributed to intraindividual variation of CYP activities. The correlations and similarities among 10 CYP activities were analyzed, and it was found that the highest correlation and similarity for Vmax, Km, and CLint occurred between CYP2C9 and 2C8, CYP2C9 and 1A2, and CYP2C9 and 2C8, respectively. In conclusion, CYP activities exhibit considerable intraindividual variation, with some notable correlations, which might be helpful to comprehensively understand the internal connection among CYP activities and to guide the rational use of drugs.

5-Hydroxymethylome in Circulating Cell-free DNA as A Potential Biomarker for Non-small-cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls (mean decrease accuracy >3.2, P < 3.68E-19) using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient's cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.

The effect of fertility stress on endometrial and subendometrial blood flow among infertile women.

BACKGROUND: To investigate the effect of fertility stress on endometrial and subendometrial blood flow among infertile women. METHODS: This case-control study was conducted in The First Affiliated Hospital of Zhengzhou University. The fertility problem inventory (FPI) was adopted to evaluate fertility stress. Three-dimensional power Doppler ultrasonography (3D PD-US) was performed during the proliferative phase of the menstrual cycle (days 5-11) to measure endometrial thickness, pattern, endometrial and subendometrial volume (V), the vascularization index (VI), the flow index (FI) and the vascularization-FI (VFI) index. Then, 300 infertile women were separated into two groups (high-score group and low-score group) based on total FPI scores and 80 healthy women were selected as controls. RESULTS: No differences were found among all three groups with regard to general characteristics, endometrial thickness, pattern, endometrial and subendometrial V, VI and VFI. The endometrial and subendometrial FIs associated with different stress levels significantly differed among the three groups (F = 33.95, P < 0.001; F = 44.79, P < 0.001, respectively). The endometrial and subendometrial FIs in the control group were significantly higher than those in the high-score group and low-score groups. The endometrial and subendometrial FIs in the low-score group were significantly higher than those in the high-score group. The total FPI score was closely related to the endometrial and subendometrial FIs (r = -0.304, P < 0.001; r = -0.407, P < 0.001, respectively). CONCLUSION: Fertility stress was associated with endometrial and subendometrial flow index. Whether fertility stress might affect pregnancy outcome by reducing endometrial and subendometrial blood flow requires further research.