ESCCPred: a machine learning model for diagnostic prediction of early esophageal squamous cell carcinoma using autoantibody profiles.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with no clinically ideal biomarkers for early diagnosis. The objective of this study was to develop and validate a user-friendly diagnostic tool for early ESCC detection. METHODS: The study encompassed three phases: discovery, verification, and validation, comprising a total of 1309 individuals. Serum autoantibodies were profiled using the HuProtTM human proteome microarray, and autoantibody levels were measured using the enzyme-linked immunosorbent assay (ELISA). Twelve machine learning algorithms were employed to construct diagnostic models, and evaluated using the area under the receiver operating characteristic curve (AUC). The model application was facilitated through R Shiny, providing a graphical interface. RESULTS: Thirteen autoantibodies targeting TAAs (CAST, FAM131A, GABPA, HDAC1, HDGFL1, HSF1, ISM2, PTMS, RNF219, SMARCE1, SNAP25, SRPK2, and ZPR1) were identified in the discovery phase. Subsequent verification and validation phases identified five TAAbs (anti-CAST, anti-HDAC1, anti-HSF1, anti-PTMS, and anti-ZPR1) that exhibited significant differences between ESCC and control subjects (P < 0.05). The support vector machine (SVM) model demonstrated robust performance, with AUCs of 0.86 (95% CI: 0.82-0.89) in the training set and 0.83 (95% CI: 0.78-0.88) in the test set. For early-stage ESCC, the SVM model achieved AUCs of 0.83 (95% CI: 0.79-0.88) in the training set and 0.83 (95% CI: 0.77-0.90) in the test set. Notably, promising results were observed for high-grade intraepithelial neoplasia, with an AUC of 0.87 (95% CI: 0.77-0.98). The web-based implementation of the early ESCC diagnostic tool is publicly accessible at https://litdong.shinyapps.io/ESCCPred/ . CONCLUSION: This study provides a promising and easy-to-use diagnostic prediction model for early ESCC detection. It holds promise for improving early detection strategies and has potential implications for public health.

Anti-BIRC5 autoantibody serves as a valuable biomarker for diagnosing AFP-negative hepatocellular carcinoma.

Background: Autoantibodies targeting tumor-associated antigens (TAAbs) have emerged as promising biomarkers for early cancer detection. This research aimed to assess the diagnostic capacity of anti-BIRC5 autoantibody in detecting AFP-negative hepatocellular carcinoma (ANHCC). Methods: This research was carried out in three stages (discovery phase, validation phase, and evaluation phase) and included a total of 744 participants. Firstly, the anti-BIRC5 autoantibody was discovered using protein microarray, exhibiting a higher positive rate in ANHCC samples (ANHCCs) compared to normal control samples (NCs). Secondly, the anti-BIRC5 autoantibody was validated through enzyme-linked immunosorbent assay (ELISA) in 85 ANHCCs and 85 NCs from two clinical centers (Zhengzhou and Nanchang). Lastly, the diagnostic usefulness of the anti-BIRC5 autoantibody for hepatocellular carcinoma (HCC) was evaluated by ELISA in a cohort consisting of an additional 149 AFP-positive hepatocellular carcinoma samples (APHCCs), 95 ANHCCs and 244 NCs. The association of elevated autoantibody to high expression of BIRC5 in HCC was further explored by the database from prognosis, immune infiltration, DNA methylation, and gene mutation level. Results: In the validation phase, the area under the ROC curve (AUC) of anti-BIRC5 autoantibody to distinguish ANHCCs from NCs in Zhengzhou and Nanchang centers was 0.733 and 0.745, respectively. In the evaluation phase, the AUCs of anti-BIRC5 autoantibody for identifying ANHCCs and HCCs from NCs were 0.738 and 0.726, respectively. Furthermore, when combined with AFP, the AUC for identifying HCCs from NCs increased to 0.914 with a sensitivity of 77.5% and specificity of 91.8%. High expression of BIRC5 gene is not only correlated with poor prognosis of HCCs, but also significantly associated with infiltration of immune cells, DNA methylation, and gene mutation. Conclusion: The findings suggest that the anti-BIRC5 autoantibody could serve as a potential biomarker for ANHCC, in addition to its supplementary role alongside AFP in the diagnosis of HCC. Next, we can carry out specific verification and explore the function of anti-BIRC5 autoantibody in the occurrence and development of HCC.

CCDC12 gene methylation in peripheral blood as a potential biomarker for breast cancer detection.

BACKGROUND: Aberrant DNA methylation has been identified as biomarkers for breast cancer detection. Coiled-coil domain containing 12 gene (CCDC12) implicated in tumorigenesis. This study aims to investigate the potential of blood-based CCDC12 methylation for breast cancer detection. METHODS: DNA methylation level of CpG sites (Cytosine-phosphate Guanine dinucleotides) in CCDC12 gene was measured by mass spectrometry in 255 breast cancer patients, 155 patients with benign breast nodules and 302 healthy controls. The association between CCDC12 methylation and breast cancer risk was evaluated by logistic regression and receiver operating characteristic curve analysis. RESULTS: A total of eleven CpG sites were analyzed. The CCDC12 methylation levels were higher in breast cancer patients. Compared to the lowest tertile of methylation level in CpG_6,7, CpG_10 and CpG_11, the highest quartile was associated with 82, 91 and 95% increased breast cancer risk, respectively. The CCDC12 methylation levels were associated with estrogen receptor (ER) and human epidermal growth factor 2 (HER2) status. In ER-negative and HER2-positive (ER-/HER2+) breast cancer subtype, the combination of four sites CpG_2, CpG_5, CpG_6,7 and CpG_11 methylation levels could distinguish ER-/HER2+ breast cancer from the controls (AUC = 0.727). CONCLUSION: The hypermethylation levels of CCDC12 in peripheral blood could be used for breast cancer detection.

Breast cancer detection could be facilitated by novel blood-based DNA methylation biomarkers.The methylation levels of CpG sites in CCDC12 were higher in breast cancer than those in controls.The combination of four sites CpG_2, CpG_5, CpG_6,7 and CpG_11 methylation levels could distinguish ER-/HER2+ breast cancer subtype from the controls.

SPP1 represents a therapeutic target that promotes the progression of oesophageal squamous cell carcinoma by driving M2 macrophage infiltration.

BACKGROUND: Tumour-associated macrophages (TAMs) are an important component of the tumour microenvironment (TME). However, the crosstalk between oesophageal squamous cell carcinoma (ESCC) cells and TAMs remains largely unexplored. METHODS: Clinical samples and the TCGA database were used to evaluate the relevance of SPP1 and TAM infiltration in ESCC. Mouse models were constructed to investigate the roles of macrophages educated by SPP1 in ESCC. Macrophage phenotypes were determined using qRT‒PCR and immunohistochemical staining. RNA sequencing was performed to elucidate the mechanism. RESULTS: Increasing expression of SPP1 correlated with M2-like TAM accumulation in ESCC, and they both predicted poor prognosis in the ESCC cohort. Knockdown of SPP1 significantly inhibited the infiltration of M2 TAMs in xenograft tumours. In vivo mouse model experiments showed that SPP1-mediated education of macrophages plays an essential role in the progression of ESCC. Mechanistically, SPP1 recruited macrophages and promoted M2 polarisation via CD44/PI3K/AKT signalling activation and then induced VEGFA and IL6 secretion to sustain ESCC progression. Finally, blockade of SPP1 with RNA aptamer significantly inhibited tumour growth and M2 TAM infiltration in xenograft mouse models. CONCLUSIONS: This study highlights SPP1-mediated crosstalk between ESCC cells and TAMs in ESCC. SPP1 could serve as a potential target in ESCC therapy.

Ethanolic extract of Arctium lappa leaves alleviates cerebral ischemia reperfusion-induced inflammatory injury via HDAC9-mediated NF-κB pathway.

BACKGROUND: Ischemic stroke (IS) is a major cause of mortality and disability worldwide. Inflammatory response is crucial in the pathogenesis of tissue injury in cerebral infarction. Arctium lappa leaves are traditionally used to treat IS. PURPOSES: To investigate the neuroprotective effects and molecular mechanisms of the ethanolic extract of A. lappa leaves (ALLEE) on cerebral ischemia-reperfusion (CIR). METHODS: Middle cerebral artery obstruction reperfusion (MCAO/R) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were used to evaluate ALLEE pharmacodynamics. Various methods, including neurological function, 2,3,5-triphenyltetrazolium chloride, hematoxylin and eosin, and Nissl, enzyme-linked immunosorbent, and TdT-mediated dUTP nick-end labeling assays, were used to analyze the neuroprotective effects of ALLEE in vitro and in vivo. The major chemical components and potential target genes of ALLEE were screened using network pharmacology. Molecular docking, western blotting, and immunofluorescence analyses were performed to confirm the effectiveness of the targets in related pathways. RESULTS: ALLEE exerted potent effects on the MCAO/R model by decreasing the neurological scores, infarct volumes, and pathological features (p < 0.01). Furthermore, network pharmacology results revealed that the treatment of IS with ALLEE involved the regulation of various inflammatory pathways, such as the tumor necrosis factor (TNF) and chemokine signaling pathways. ALLEE also played key roles in targeting key molecules, including nuclear factor (NF)-κBIA, NF-κB1, interleukin (IL)-6, TNF-α and IL1ß, and regulating the histone deacetylase (HDAC)-9-mediated signaling pathway. In vivo and in vitro analyses revealed that ALLEE significantly regulated the NF-κB pathway, promoted the phosphorylation activation of NF-κB P65, IκB and IKK (p < 0.01 or p < 0.05), and decreased the expression levels of the inflammatory factors, IL-1ß, IL-6 and TNF-α (p < 0.01). Moreover, ALLEE significantly decreased the expression of HDAC9 (p < 0.01) that is associated with inflammatory responses. However, HDAC9 overexpression partially reversed the neuroprotective effects of ALLEE and its suppressive effects on inflammation and phosphorylation of NF-κB (p < 0.01). CONCLUSIONS: In conclusion, our results revealed that ALLEE ameliorates MCAO/R-induced experimental CIR by modulating inflammatory responses via the inhibition of HDAC9-mediated NF-κB pathway.

Aqueous extract of fermented Eucommia ulmoides leaves alleviates hyperlipidemia by maintaining gut homeostasis and modulating metabolism in high-fat diet fed rats.

BACKGROUND: As a traditional Chinese medicinal herb, the lipid-lowing biological potential of Eucommia ulmoides leaves (EL) has been demonstrated. After fermentation, the EL have been made into various products with lipid-lowering effects and antioxidant activity. However, the anti-hyperlipidemic mechanism of fermented Eucommia ulmoides leaves (FEL) is unclear now. PURPOSE: To evaluate the effects of FEL on hyperlipidemia and investigate the mechanism based on regulating gut homeostasis and host metabolism. METHODS: Hyperlipidemia animal model in Wistar rats was established after 8 weeks high-fat diet (HFD) fed. The administered doses of aqueous extract of FEL (FELE) were 128, 256 and 512 mg/kg/d, respectively. Serum biochemical parameters detection, histopathological sections analysis, 16S rDNA sequencing of gut microbiota and untargeted fecal metabolomics analysis, were performed to determine the therapeutic effects and predict related pathways of FELE on hyperlipidemia. The changes of proteins and genes elated to lipid were detected by Immunofluorescence (IF) and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: 56 Components in FELE were identified by UPLC-MS, with organic acids, flavonoids and phenolic acids accounting for the majority. The intervention of FELE significantly reduced the body weight, lipid accumulation and the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein-cholesterol (LDL-C) in hyperlipidemia rats, while increased the level of High-density lipoprotein-cholesterol (HDL-C). Meanwhile, FELE improved the inflammatory makers and oxidative stress factors, which is tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT). These results demonstrated that FETE can effectively reduce blood lipids and alleviate inflammation and oxidative damage caused by hyperlipidemia. Mechanistically, FELE restore the homeostasis of gut microbiota by reducing the Firmicutes/Bacteroidetes ratio and increasing the abundance of probiotics, especially Lactobacillus, Rombousia, Bacteroides, Roseburia, Clostridia_UCG-014_Unclassified, while modulated metabolism through amino acid, bile acid and lipid-related metabolism pathways. In addition, the Pearson correlation analysis found that the upregulated bilirubin, threonine, dopamine and downregulated lipocholic acid, d-sphingosine were key metabolites after FELE intervention. IF and qRT-PCR analysis showed that FELE upregulated the expression of fatty acid oxidation proteins and genes (PPARα, CPT1A), bile acid synthesis and excretion proteins and genes (LXRα, CYP7A1, FXR), and downregulated the expression of adipogenic gene (SREBP-1c) by regulating gut microbiota to improve metabolism and exert a lipid-lowering effect. CONCLUSION: This work filled the lipid-lowering mechanism gap of FEL. FELE can improve HFD-induced hyperlipidemia by regulating the gut microbiota homeostasis and metabolism. Thus, FEL has the potential to develop into the novel raw material of lipid-lowering drugs.

Humoral immune response to tumor-associated antigen Ubiquilin 1 (UBQLN1) and its tumor-promoting potential in lung cancer.

BACKGROUND: This study aims to investigate the expression of UBQLN1 in lung cancer (LC) tissue and the diagnostic capability of autoantibody to UBQLN1 (anti-UBQLN1) in the detection of LC and the discrimination of pulmonary nodules (PNs). METHODS: Sera from 798 participants were used to discover and validate the level of autoantibodies via HuProt microarray and Enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was applied to establish model. Receiver operating characteristic curve (ROC) analysis was performed to evaluate the diagnostic potential. Immunohistochemistry was performed to detect UBQLN1 expression in 88 LC tissues and 88 para-tumor tissues. qRT-PCR and western blotting were performed to detect the expression of UBQLN1 at the mRNA and protein levels, respectively. Trans-well assay and cell counting kit-8 (CCK-8) was used to investigate the function of UBQLN1. RESULTS: Anti-UBQLN1 was identified with the highest fold change by protein microarray. The level of anti-UBQLN1 in LC patients was obviously higher than that in NC or patients with benign lung disease of validation cohort 1 (P<0.05). The area under the curve (AUC) of anti-UBQLN1 was 0.610 (95%CI: 0.508-0.713) while reached at 0.822 (95%CI: 0.784-0.897) when combining anti-UBQLN1 with CEA, CYFRA21-1, CA125 and three CT indicators (vascular notch sign, lobulation sign and mediastinal lymph node enlargement) in the discrimination of PNs. UBQLN1 protein was overexpressed in lung adenocarcinoma (LUAD) tissues compared to para-tumor tissues. UBQLN1 knockdown remarkably inhibited the migration, invasion and proliferation of LUAD cell lines. CONCLUSIONS: Anti-UBQLN1 might be a potential biomarker for the diagnosis of LC and the discrimination of PNs.

Signature construction and molecular subtype identification based on liver-specific genes for prediction of prognosis, immune activity, and anti-cancer drug sensitivity in hepatocellular carcinoma.

BACKGROUND: Liver specific genes (LSGs) are crucial for hepatocyte differentiation and maintaining normal liver function. A deep understanding of LSGs and their heterogeneity in hepatocellular carcinoma (HCC) is necessary to provide clues for HCC diagnosis, prognosis, and treatment. METHODS: The bulk and single-cell RNA-seq data of HCC were downloaded from TCGA, ICGC, and GEO databases. Through unsupervised cluster analysis, LSGs-based HCC subtypes were identified in TCGA-HCC samples. The prognostic effects of the subtypes were investigated with survival analyses. With GSVA and Wilcoxon test, the LSGs score, stemness score, aging score, immune score and stromal score of the samples were estimated and compared. The HCC subtype-specific genes were identified. The subtypes and their differences were validated in ICGC-HCC samples. LASSO regression analysis was used for key gene selection and risk model construction for HCC overall survival. The model performance was estimated and validated. The key genes were validated for their heterogeneities in HCC cell lines with quantitative real-time PCR and at single-cell level. Their dysregulations were investigated at protein level. Their correlations with HCC response to anti-cancer drugs were estimated in HCC cell lines. RESULTS: We identified three LSGs-based HCC subtypes with different prognosis, tumor stemness, and aging level. The C1 subtype with low LSGs score and high immune score presented a poor survival, while the C2 subtype with high LSGs score and immune score indicated an enduring survival. Although no significant survival difference between C2 and C3 HCCs was shown, the C2 HCCs presented higher immune score and stroma score. The HCC subtypes and their differences were confirmed in ICGC-HCC dataset. A five-gene prognostic signature for HCC survival was constructed. Its good performance was shown in both the training and validation datasets. The five genes presented significant heterogeneities in different HCC cell lines and hepatocyte subclusters. Their dysregulations were confirmed at protein level. Furthermore, their significant associations with HCC sensitivities to anti-cancer drugs were shown. CONCLUSIONS: LSGs-based HCC subtype classification and the five-gene risk model might provide useful clues not only for HCC stratification and risk prediction, but also for the development of more personalized therapies for effective HCC treatment.

The association between circulating 25-hydroxyvitamin D and pancreatic cancer: a systematic review and meta-analysis of observational studies.

PURPOSE: The relationship between circulating 25-hydroxyvitamin D [25(OH)D] and pancreatic cancer has been well studied but remains unclear. The purpose of this study was to elucidate the association between circulating 25(OH)D and pancreatic cancer by using a meta-analytic approach. METHODS: PubMed, Embase, and Wed of Science databases were searched through October 15, 2022. A random or fixed-effects model was used to estimate the pooled odds ratio (OR), risk ratio (RR), hazard ratio (HR) and their 95% confidence intervals (CIs). RESULTS: A total of 16 studies including 529,917 participants met the inclusion criteria, of which 10 reported incidence and 6 reported mortality. For the highest versus lowest categories of circulating 25(OH)D, the pooled OR of pancreatic cancer incidence in case-control studies was 0.98 (95% CI 0.69-1.27), and the pooled HRs of pancreatic cancer mortality in cohort and case-control studies were 0.64 (95% CI 0.45-0.82) and 0.78 (95% CI 0.62-0.95), respectively. The leave-one-out sensitivity analyses found no outliers and Galbraith plots indicated no substantial heterogeneity. CONCLUSION: Evidence from this meta-analysis suggested that high circulating 25(OH)D levels may be associated with decreased mortality but not incidence of pancreatic cancer. Our findings may provide some clues for the treatment of pancreatic cancer and remind us to be cautious about widespread vitamin D supplementation for the prevention of pancreatic cancer.

Comparison of anterior cervical diskectomy with fusion (ACDF) and laminoplasty treating multilevel cervical spondylotic myelopathy with developmental canal stenosis: a retrospective study.

PURPOSE: To evaluate clinical effectiveness and radiologic results of anterior cervical diskectomy with fusion (ACDF) comparing with laminoplasty (LP) in treating multilevel cervical spondylotic myelopathy (MCSM) with developmental canal stenosis (DCS). METHODS: This was a retrospective analysis of 41 patients who had MCSM with DCS treated with ACDF or LP from December 2018 to April 2023. Patients were split into ACDF and LP groups for comparison, and patients were further separated into subgroups based on whether or not a reserving canal space was present. The operation time, hemoglobin, hospital stay, modified Japanese Orthopaedic Association (mJOA) score, and visual analog scale (VAS) score were used to assess clinical efficacy. The C2-C7 Cobb angle, C2-C7 sagittal vertical axis, T1 slope, and cervical range of motion were applied to evaluate imaging changes. RESULTS: Of the 41 patients, 19 received ACDF, and 22 received LP. At the final follow-up, both groups' mJOA scores significantly improved, and the intercomparison showed no differences; the VAS score was much lower in the ACDF group but remained unchanged in the LP group. At the final follow-up, the C2-C7 Cobb angle and T1 slope had significantly increased in the ACDF group, while the LP group showed no change; the cervical range of motion had significantly decreased in both groups, with the ACDF group exhibiting a more marked reduction. Within the ACDF subgroup, there was no postoperative symptom improvement for those with reserving space, whereas there was postoperative symptom resolution for those with non-reserving space; however, postoperative symptom in the LP subgroup was resolved. CONCLUSIONS: Both ACDF and LP were efficacious for MCSM patients with DCS. While ACDF could improve cervical lordosis and alleviate neck pain more effectively, it can also result in cervical sagittal imbalance and decreased mobility. Furthermore, the recovery from LP was superior to that from ACDF for patients with reserving space. In contrast, the recovery from both decompression techniques was comparable for individuals in non-reserving space.

Blood FOLR3 methylation dysregulations and heterogeneity in non-small lung cancer highlight its strong associations with lung squamous carcinoma.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers. Early detection is crucial to reduce lung cancer-related mortality. Aberrant DNA methylation occurs early during carcinogenesis and can be detected in blood. It is essential to investigate the dysregulated blood methylation markers for early diagnosis of NSCLC. METHODS: NSCLC-associated methylation gene folate receptor gamma (FOLR3) was selected from an Illumina 850K array analysis of peripheral blood samples. Mass spectrometry was used for validation in two independent case-control studies (validation I: n = 2548; validation II: n = 3866). Patients with lung squamous carcinoma (LUSC) or lung adenocarcinoma (LUAD), normal controls (NCs) and benign pulmonary nodule (BPN) cases were included. FOLR3 methylations were compared among different populations. Their associations with NSCLC clinical features were investigated. Receiver operating characteristic analyses, Kruskal-Wallis test, Wilcoxon test, logistics regression analysis and nomogram analysis were performed. RESULTS: Two CpG sites (CpG_1 and CpG_2) of FOLR3 was significantly lower methylated in NSCLC patients than NCs in the discovery round. In the two validations, both LUSC and LUAD patients presented significant FOLR3 hypomethylations. LUSC patients were highlighted to have significantly lower methylation levels of CpG_1 and CpG_2 than BPN cases and LUAD patients. Both in the two validations, CpG_1 methylation and CpG_2 methylation could discriminate LUSC from NCs well, with areas under the curve (AUCs) of 0.818 and 0.832 in validation I, and 0.789 and 0.780 in validation II. They could also differentiate LUAD from NCs, but with lower efficiency. CpG_1 and CpG_2 methylations could also discriminate LUSC from BPNs well individually in the two validations. With the combined dataset of two validations, the independent associations of age, gender, and FOLR3 methylation with LUSC and LUAD risk were shown and the age-gender-CpG_1 signature could discriminate LUSC and LUAD from NCs and BPNs, with higher efficiency for LUSC. CONCLUSIONS: Blood-based FOLR3 hypomethylation was shown in LUSC and LUAD. FOLR3 methylation heterogeneity between LUSC and LUAD highlighted its stronger associations with LUSC. FOLR3 methylation and the age-gender-CpG_1 signature might be novel diagnostic markers for the early detection of NSCLC, especially for LUSC.

An ethanolic extract of Arctium lappa L. leaves ameliorates experimental atherosclerosis by modulating lipid metabolism and inflammatory responses through PI3K/Akt and NF-κB singnaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS), a lipid-induced inflammatory condition of the arteries, is a primary contributor to atherosclerotic cardiovascular diseases including stroke. Arctium lappa L. leaf (ALL), an edible and medicinal herb in China, has been documented and commonly used for treating stroke since the ancient times. However, the elucidations on its anti-AS effects and molecular mechanism remain insufficient. AIM OF THE STUDY: To investigate the AS-ameliorating effects and the underlying mechanism of action of an ethanolic extract of leaves of Arctium lappa L. (ALLE). MATERIALS AND METHODS: ALLE was reflux extracted using with 70% ethanol. An HPLC method was established to monitor the quality of ALLE. High fat diet (HFD) and vitamin D3-induced experimental AS in rats were used to determine the in vivo effects; and oxidized low-density lipoprotein-induced RAW264.7 macrophage foam cells were used for in vitro assays. Simvatatin was used as positive control. Biochemical assays were implemented to ascertain the secretions of lipids and pro-inflammatory mediators. Haematoxylin-eosin (H&E) and Oil red O stains were employed to assess histopathological alterations and lipid accumulation conditions, respectively. CCK-8 assays were used to measure cytotoxicity. Immunoblotting assay was conducted to measure protein levels. RESULTS: ALLE treatment significantly ameliorated lipid deposition and histological abnormalities of aortas and livers in AS rats; improved the imbalances of serum lipids including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C); notably attenuated serum concentrations of inflammation-associated cytokines/molecules including TNF-α, IL-6, IL-1ß, VCAM-1, ICAM-1and MMP-9. Mechanistic studies demonstrated that ALLE suppressed the phosphorylation/activation of PI3K, Akt and NF-κB in AS rat aortas and in cultured foam cells. Additionally, the PI3K agonist 740Y-P notably reversed the in vitro inhibitory effects of ALLE on lipid deposition, productions of TC, TNF-α and IL-6, and protein levels of molecules of PI3K/Akt and NF-κB singnaling pathways. CONCLUSIONS: ALLE ameliorates HFD- and vitamin D3-induced experimental AS by modulating lipid metabolism and inflammatory responses, and underlying mechanisms involves inhibition of the PI3K/Akt and NF-κB singnaling pathways. The findings of this study provide scientific justifications for the traditional application of ALL in managing atherosclerotic diseases.

ZPR1 is an immunodiagnostic biomarker and promotes tumor progression in esophageal squamous cell carcinoma.

To evaluate the potential of zinc finger protein 1 (ZPR1) as a diagnostic biomarker and explore the underlying role for esophageal squamous cell carcinoma (ESCC). A human proteome microarray was customized to identify anti-ZPR1 autoantibody, and enzyme-linked immunosorbent assay (ELISA) was adopted to assess the diagnostic performance of anti-ZPR1 autoantibody in 294 patients with ESCC and 294 normal controls. The expression of ZPR1 protein was measured by immunohistochemistry. The effect of ZPR1 on the proliferation, migration, and invasion of ESCC cells was investigated through CCK-8, wound healing, and Transwell assays. The expression level of anti-ZPR1 autoantibody (fold change = 2.77) in ESCC patients was higher than that in normal controls. The receiver operating characteristic (ROC) analysis manifested anti-ZPR1 autoantibody achieved area under the ROC curve (AUC) of 0.726 and 0.734 to distinguish ESCC from normal controls with sensitivity of 50.0% and 42.3%, and specificity of 91.0% and 92.0% in the test group and validation group, respectively. The positive rate of ZPR1 protein was significantly higher in ESCC tissues (75.5%, 80/106) than paracancerous tissues (9.4%, 5/53). Compared with the human normal esophageal cell line, the expression level of ZPR1 mRNA and protein in ESCC lines (KYSE150, Eca109, and TE1) had an increased trend. The knockdown or overexpression of ZPR1 reduced and enhanced the proliferation, migration, and invasion of ESCC cell, respectively. ZPR1 was a potential immunodiagnostic biomarker for noninvasive detection and could be a promotional factor in tumor progression of ESCC.

Integrative Multianalytical Model Based on Novel Plasma Protein Biomarkers for Distinguishing Lung Adenocarcinoma and Benign Pulmonary Nodules.

Given the pressing clinical problem of making a decision in diagnosis for subjects with pulmonary nodules, we aimed to discover novel plasma protein biomarkers for lung adenocarcinoma (LUAD) and benign pulmonary nodules (BPNs) and then develop an integrative multianalytical model to guide the clinical management of LUAD and BPN patients. Through label-free quantitative plasma proteomic analysis (data are available via ProteomeXchange with identifier PXD046731), 12 differentially expressed proteins (DEPs) in LUAD and BPN were screened. The diagnostic abilities of DEPs were validated in two independent validation cohorts. The results showed that the levels of three candidate proteins (PRDX2, PON1, and APOC3) were lower in the plasma of LUAD than in BPN. The three candidate proteins were combined with three promising computed tomography indicators (spiculation, vascular notch sign, and lobulation) and three traditional markers (CEA, CA125, and CYFRA21-1) to construct an integrative multianalytical model, which was effective in distinguishing LUAD from BPN, with an AUC of 0.904, a sensitivity of 81.44%, and a specificity of 90.14%. Moreover, the model possessed impressive diagnostic performance between early LUADs and BPNs, with the AUC, sensitivity, specificity, and accuracy of 0.868, 65.63%, 90.14%, and 82.52%, respectively. This model may be a useful auxiliary diagnostic tool for LUAD and BPN by achieving a better balance of sensitivity and specificity.

Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma.

Purpose: This study was based on hepatocellular carcinoma (HCC) patients of early-stage to explore the diagnostic capability and possible production causes of anti-GNAS autoantibody. Methods: We evaluated the frequency of anti-GNAS autoantibody in sera from patients with early-stage HCC by enzyme-linked immunosorbent assay (ELISA) and the expression of GNAS protein in early-stage HCC tissues by immunohistochemistry. Western blotting (WB) and real-time polymerase chain reaction (RT-PCR) were utilized to examine the expressions of GNAS protein and mRNA in cell lines. GEO and International Cancer Genome Consortium (ICGC) databases were inquired to explore mRNA expression and mutation of GNAS in HCC tissues. Results: The positive rates of anti-GNAS autoantibody in HCC patients at clinical stage I (78.1 %) and clinical stage II (57.1 %) were all significantly higher than that in healthy control (20 %). There was also a significant difference in GNAS protein expression between HCC and its adjacent normal liver tissues. The results from WB and RT-PCR showed a significant difference at the mRNA level but no statistical difference at the protein level between HCC and normal liver cell lines. The difference in mRNA level between HCC and adjacent normal liver tissues was verified to be significant. Furthermore, the ICGC database demonstrated a 10.6 % mutation frequency for GNAS in HCC patients. Conclusion: The coordination of elevated anti-GNAS autoantibody, high expression of GNAS in the mRNA and protein levels in HCC, and high frequency of GNAS mutation indicates that anti-GNAS autoantibody may be used as an early indicator of HCC.

Novel Biomarker miRNA-625-3p Promotes Cell Proliferation and Metastasis of Lung Adenocarcinoma by Targeting KLF9.

BACKGROUND: MicroRNAs (miRNAs) are emerging as potential blood-based biomarkers involved in various types of carcinogenesis, including lung adenocarcinoma (LUAD). MATERIALS AND METHODS: In the present study, microarray was used to screen 2,549 miRNAs in serum samples from seven patients with LUAD and seven from healthy controls. Quantitative real-time polymerase chain reaction was used to validate the expression of miRNA in serum samples from 30 patients with LUAD and 30 heathy individuals. The area under the receiver operating characteristic curve was determined to evaluate the diagnostic capability of miR-625-3p. Cell counting kit-8 assay and Transwell assays were used to explore cell proliferation, migration and invasion. Bioinformatics prediction was applied in the search for the target genes of miR-625-3p. Quantitative real-time polymerase chain reaction, western blot and dual luciferase assay were used to validate target genes of miR-625-3p. A xenograft tumor model was established to evaluate cell proliferation in vivo. RESULTS: miR-625-3p was the miRNA most highly expressed in serum samples from patients with LUAD according to microarray analysis, this finding was verified in sera from an independent cohort, as well as in tissues based on The Cancer Genome Atlas database. Serum miR-625-3p provided a high diagnostic accuracy for LUAD (area under the curve=0.790, 95% confidence interval=0.6640-0.9152). Functionally, miR-625-3p promoted LUAD cell proliferation, migration and invasion both in vivo and in vitro. Mechanistically, we found miR-625-3p promoted cell proliferation and metastasis of LUAD by directly targeting KLF transcription factor 9 (Kruppel-like factor 9, KLF9). CONCLUSION: Our study identified that miR-625-3p plays an oncogenic role in LUAD, targeting KLF9. miR-625-3p might be a potential novel diagnostic biomarker and target for LUAD therapy.

Hypomethylation of DYRK4 in peripheral blood is associated with increased lung cancer risk.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. It is urgent to identify new biomarkers for the early detection of LC. DNA methylation in peripheral blood has been reported to be associated with cancers. We conducted two independent case-control studies and a nested case-control study (168 LC cases and 167 controls in study Ⅰ, 677 LC cases and 833 controls in study Ⅱ, 147 precancers and 21 controls in the nested case-control study). The methylation levels of DYRK4 CpG sites were measured using mass spectrometry and their correlations with LC were analyzed by logistic regression and nonparametric tests. Bonferroni correction was used for the multiple comparisons. LC-related decreased DYRK4 methylation was discovered in Study I and validated in Study II (the odds ratios [ORs] for the lowest vs. highest quartile of all three DYRK4 CpG sites ranged from 1.64 to 2.09, all p < 0.001). Combining the two studies, hypomethylation of DYRK4 was observed in stage I cases (ORs per -10% methylation ranged from 1.16 to 1.38, all p < 5.9E-04), and could be enhanced by male gender (ORs ranged from 1.77 to 4.17 via interquartile analyses, all p < 0.017). Hypomethylation of DYRK4_A_CpG_2 was significantly correlated with tumor size, length, and stage (p = 0.034, 0.002, and 0.002, respectively) in LC cases. Our study disclosed the association between DYRK4 hypomethylation in peripheral blood and LC, suggesting the feasibility of blood-based DNA methylation as new biomarker for LC detection.

Identification of Chemical Constituents in Blumea balsamifera Using UPLC-Q-Orbitrap HRMS and Evaluation of Their Antioxidant Activities.

Blumea balsamifera (L.) DC., a perennial herb in the Asteraceae family native to China and Southeast Asia, has a notable history of medicinal use due to its pharmacological properties. Using UPLC-Q-Orbitrap HRMS techniques, we systematically investigated the chemical constituents of this plant. A total of 31 constituents were identified, of which 14 were flavonoid compounds. Significantly, 18 of these compounds were identified in B. balsamifera for the first time. Furthermore, the mass spectrometry fragmentation patterns of significant chemical constituents identified in B. balsamifera were analyzed, providing important insights into their structural characteristics. The in vitro antioxidative potential of the methanol extract of B. balsamifera was assessed using DPPH and ABTS free-radical-scavenging assays, total antioxidative capacity, and reducing power. The antioxidative activity exhibited a direct correlation with the mass concentration of the extract, with IC50 values of 105.1 ± 0.503 µg/mL and 12.49 ± 0.341 µg/mL for DPPH and ABTS, respectively. For total antioxidant capacity, the absorbance was 0.454 ± 0.009 at 400 µg/mL. In addition, the reducing power was 1.099 ± 0.03 at 2000 µg/mL. This study affirms that UPLC-Q-Orbitrap HRMS can effectively discern the chemical constituents in B. balsamifera, primarily its flavonoid compounds, and substantiates its antioxidative properties. This underscores its potential utility as a natural antioxidant in the food, pharmaceutical, and cosmetics sectors. This research provides a valuable theoretical basis and reference value for the comprehensive development and utilization of B. balsamifera and expands our understanding of this medicinally valuable plant.

The association between CTSZ methylation in peripheral blood and breast cancer in Chinese women.

Purpose: Previous studies have shown that DNA methylation in peripheral blood may be associated with breast cancer (BC). To explore the association between the methylation level of the Cathepsin Z (CTSZ) gene in peripheral blood and BC, we conducted a case-control study in the Chinese population. Methods: Peripheral blood samples were collected from 567 BC cases, 635 healthy controls, and 303 benign breast disease (BBD) cases. DNA extraction and bisulfite-specific PCR amplification were performed for all samples. The methylation levels of seven sites of the CTSZ gene were quantitatively determined by Mass spectrometry. The odds ratios (ORs) of CpG sites were evaluated for BC risk using per 10% reduction and quartiles analyses by logistic regression. Results: Our analysis showed that five out of the seven CpG sites exhibited significant associations with hypomethylation of CTSZ and BC, compared to healthy controls. The highest OR was for Q2 of CTSZ_CpG_1 (OR: 1.62, P=0.006), particularly for early-stage breast cancer in the case of per 10% reduction of CTSZ_CpG_1 (OR: 1.20, P=0.003). We also found that per 10% reduction of CTSZ_CpG_5 (OR: 1.39, P=0.004) and CTSZ_CpG_7,8 (OR: 1.35, P=0.005) were associated with increased BC risk. Our study also revealed that four out of seven CpG sites were linked to increased BC risk in women under 50 years of age, compared to healthy controls. The highest OR was for per 10% reduction of CTSZ_CpG_1 (OR: 1.47, P<0.001). Additionally, we found that BC exhibited lower methylation levels than BBD at CTSZ_CpG_4 (OR for Q1: 2.18, P<0.001) and CTSZ_CpG_7,8 (OR for Q1: 2.01, P=0.001). Furthermore, we observed a correlation between methylation levels and tumor stage, ER, and HER2 status in BC patients. Conclusion: Overall, our findings suggest that altered CTSZ methylation levels in peripheral blood may be associated with breast cancer, particularly in young women, and may serve as a potential biomarker for early-stage BC.

LncRNA XIST promotes adjuvant-induced arthritis by increasing the expression of YY1 via miR-34a-5p.

Objectives: This study aims to explore the mechanism by which long non-coding ribonucleic acids (lncRNA) X-inactive specific transcript (XIST) affects the progression of adjuvant-induced arthritis (AIA). Materials and methods: Freund's complete adjuvant was used to induce arthritis in rats. The polyarthritis, spleen and thymus indexes were calculated to evaluate AIA. Hematoxylin-eosin (H&E) staining was used to reveal the pathological changes in the synovium of AIA rats. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-8 in the synovial fluid of AIA rats. The cell continuing kit (CCK)-8, flow cytometry, and Transwell assays were used to assess the proliferation, apoptosis, migration and invasion of transfected fibroblast-like synoviocytes (FLS) isolated from AIA rats (AIA-FLS). Dual-luciferase reporter assay was performed to verify the binding sites between XIST and miR-34b-5p or between YY1 mRNA and miR-34b-5p. Results: The XIST and YY1 were highly expressed, and miR-34a-5p was lowly expressed in the synovium of AIA rats and in AIA-FLS. Silencing of XIST impaired the function of AIA-FLS in vitro and inhibited the progression of AIA in vivo. The XIST promoted the expression of YY1 by competitively binding to miR-34a-5p. Inhibition of miR-34a-5p strengthened the function of AIA-FLS by upregulating XIST and YY1. Conclusion: The XIST controls the function of AIA-FLS and may promote the progression of rheumatoid arthritis via the miR-34a-5p/YY1 axis.