Uncovering the effect and mechanism of Jiawei Xiaoyao Wan in treating breast cancer complicated with depression based on network pharmacology and experimental analysis.

BACKGROUND: Depression is a clinically common co-morbidity in breast cancer cases that brings negative outcomes on quality of life and potentially survival. Jiawei Xiaoyao Wan (JXW) is widely used in treating breast cancer and depressive disorder, but its potential pharmacological mechanisms remain elusive. PURPOSE: We aimed to explore the dual therapeutic effects and mechanisms of JXW acting on breast cancer complicated with depression (BCCD) by network pharmacology and in vivo experimental verification. METHODS: The chemical constituents of JXW were characterized using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF/MS). The targets related to constituents of JXW were predicted by the TCMSP and Swiss Target Prediction databases, and targets of breast cancer and depression were screened by the GeneCards and OMIM databases. Gene Ontology annotation and KEGG enrichment analysis were performed with the DAVID database. Ultimately, a BCCD mouse model induced by chronic restraint stress (CRS) was used to explore therapeutic effects and mechanisms of JXW against BCCD. The efficacy of JXW in the treatment of BCCD was evaluated based on behavioral tests, tumor volume and weight, and pathological examination. Additionally, the underlying mechanisms were explored by measuring the levels of neurotransmitter and inflammatory factors, as well as detecting the expression of genes or proteins associated with candidate targets and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway through RT-PCR, western blotting, and immunohistochemistry. RESULTS: Totals of 108 components were identified in JXW using LC-Q-TOF/MS. By network pharmacology analysis, 714 compound targets of JXW, 2114 breast cancer targets, 1122 depression targets, and 98 overlapping proteins were obtained. PPI network and KEGG analysis implied that TP53, ESR1, VEGFA, AKT1, IL6, TNF, EGFR and the JAK/STAT pathway might be the potential targets of JXW in treating BCCD. In vivo experiments indicated that JXW significantly ameliorated depressive symptoms and tumor progression in BCCD mice. Further mechanistic studies showed that JXW could reduce the levels of inflammatory factors, increase 5-HT level, and regulate mRNA expression levels of TP53, VEGFA, AKT1, IL6, TNF, and EGFR targets. Moreover, the expression levels of proteins related to the JAK2/STAT3 signaling pathway in BCCD mice were effectively regulated by JXW. CONCLUSION: JXW exerts dual therapeutic effects in a BCCD mouse via multiple targets. The underlying mechanisms might be associated with regulating the levels of neurotransmitter and inflammatory factors; more importantly, the JAK2/STAT3 pathway plays a significant role in this process.

Identification of m6A-Related lncRNAs Associated With Prognoses and Immune Responses in Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML) remains the most common type of hematopoietic malignancy in adults and has an unfavorable outcome. Herein, we aimed to construct an N6-methylandenosine (m6A)-related long noncoding RNAs (lncRNAs) signature to accurately predict the prognosis of patients with AML using the data downloaded from The Cancer Genome Atlas (TCGA) database. Methods: The RNA-seq and clinical data were obtained from the TCGA AML cohort. First, Pearson correlation analysis was performed to identify the m6A-related lncRNAs. Next, univariate Cox regression analysis was used to determine the candidate lncRNAs with prognostic value. Then, feature selection was carried out by Least absolute shrinkage and selection operator (LASSO) analysis, and seven eligible m6A-related lncRNAs were included to construct the prognostic risk signature. Kaplan-Meier and receiver operating characteristic (ROC) curve analyses were performed to evaluate the predictive capacity of the risk signature both in the training and testing datasets. A nomogram was used to predict 1-year, 2-year, and 3-year overall survival (OS) of AML patients. Next, the expression levels of lncRNAs in the signature were validated in AML samples by qRT-PCR. Functional enrichment analyses were carried out to identify probable biological processes and cellular pathways. The ceRNA network was developed to explore the downstream targets and mechanisms of m6A-related lncRNAs in AML. Results: Seven m6A-related lncRNAs were identified as a prognostic signature. The low-risk group hold significantly prolonged OS. The nomogram showed excellent accuracy of the signature for predicting 1-year, 2-year and 3-year OS (AUC = 0.769, 0.820, and 0.800, respectively). Moreover, the risk scores were significantly correlated with enrichment in cancer hallmark- and malignancy-related pathways and immunotherapy response in AML patients. Conclusion: We developed and validated a novel risk signature with m6A-related lncRNAs which could predict prognosis accurately and reflect the immunotherapy response in AML patients.

Absolute protein assay for the simultaneous quantification of two epoxide hydrolases in rats by mass spectrometry-based targeted proteomics.

Epoxide hydrolases catalyze the hydrolysis of both exogenous and endogenous epoxides to the corresponding vicinal diols by adding water. Microsomal and soluble epoxide hydrolase are two main mammalian enzymes that have been intensely characterized. The purpose of this investigation was to develop and validate a proteomics assay allowing the simultaneous quantification of microsomal and soluble epoxide hydrolase in rats. Protein quantification was realized through targeted proteomics using liquid chromatography with tandem mass spectrometry for the determination of trypsin-specific surrogate peptides after digestion. Stable isotope-labeled peptides were used as the internal standards. The chromatography of the surrogate peptides was performed on an Agilent SB C18 column (100 mm × 4.6 mm, 1.8 µm) with gradient elution. Acetonitrile containing 0.1% formic acid and 0.1% formic acid aqueous solution were used as mobile phases. A multiple reaction monitoring method in a positive ionization mode was used for the simultaneous detection of the peptides. The method was validated concerning the specificity, linearity, within-day and between-day accuracy and precision, matrix effect, stability, and digestion efficiency. The developed assay was successfully used to quantify the protein levels of microsomal and soluble epoxide hydrolase in rat liver, kidney, and heart S9 samples.

miR-646/TET1 mediated demethylation of IRX1 promoter upregulates HIST2H2BE and promotes the progression of invasive ductal carcinoma.

BACKGROUND: This study aimed to explore role of miR-646 in breast IDC. METHODS: miR-646, TET1, IRX1, and HIST2H2BE expression was detected by RT-qPCR and/or Western blot analysis. The methylation status of IRX1 promoter region was evaluated by methylation specific PCR. ChIP assay was used to determine the enrichment of TET1 at IRX1 promoter region. Loss- and gain-of functions were performed to determine the roles of miR-646, TET1, IRX1, and HIST2H2BE in cell proliferation, migration, invasion, and apoptosis. The tumor growth, volume, weight, and apoptosis status were measured. RESULTS: miR-646 was upregulated while TET1 was downregulated in IDC tissues. miR-646 targeted TET1. Downregulated TET1 impairs demethylation of IRX1 promoter region resulting in reduced expression of IRX1, which subsequently leads to upregulation of HIST2H2BE in IDC. Consequently, elevated HIST2H2BE promotes progression of IDC. CONCLUSION: Our study has demonstrated that miR-646 facilitates the tumorigenesis of IDC via regulating TET1/IRX1/HIST2H2BE axis.