Erratum: CircZCCHC2 decreases pirarubicin sensitivity and promotes triple-negative breast cancer development via the miR-1200/TPR axis.

[This corrects the article DOI: 10.1016/j.isci.2024.109057.].

Rapid determination of glyphosate and glufosinate in human blood by probe electrospray ionization tandem mass spectrometry.

In forensic science, glyphosate (GLYP) and glufosinate (GLUF), a class of non-selective broad-spectrum herbicides, have been frequently encountered in many fatal poisoning and suicide cases due to their widespread availability. Therefore, it is essential to develop an effective method for detecting these compounds. Some conventional methods, such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), have been reported to detect these compounds. However, these methods are not ideal for their time-consuming and non-sensitive feature. Herein, probe electrospray ionization (PESI) tandem mass spectrometry (MS/MS), a fast and sensitive technique, was applied for the determination of GLYP and GLUF in human blood, which can obtain analytical results within 0.5 min without derivatization and chromatographic separation. After protein precipitation of blood samples, the supernatant was mixed with isopropanol and ultra-pure water (1:1 v/v). Then, 8 µL of the mixture was introduced into the plastic sample plate for PESI-MS/MS analysis. The limits of detection (LODs) of the method were 0.50 µg/mL and 0.25 µg/mL for two analytes, and the limits of quantitation (LOQs) were both 1.00 µg/mL, which are higher than the concentration of reported poisoning and fatal cases. In the linear range of 1-500 µg/mL, the regression coefficients (r2) for GLYP and GLUF were over 0.99. The matrix effects ranged from 94.8 % to 119.5 %, and the biases were below 4.3 %. The recoveries ranged between 84.8 % and 107.4 %, and the biases were below 7.6 %. Meanwhile, the method was effectively utilized to detect and quantify the blood, urine, and other samples. Consequently, the results suggest that PESI-MS/MS is a straightforward, fast, and sensitive method for detecting GLUF and GLYP in forensics. In the future, PESI-MS/MS will become an indispensable technique for polar substances in grassroots units of public security where rapid detection is essential.

CircZCCHC2 decreases pirarubicin sensitivity and promotes triple-negative breast cancer development via the miR-1200/TPR axis.

Triple-negative breast cancer (TNBC) has attracted attention due to its poor prognosis and limited treatment options. The mechanisms underlying the association between circular RNAs (circRNAs) and the occurrence and development of TNBC remain unclear. CircZCCHC2 is observed to be upregulated in TNBC cells, tissues, and plasma exosomes. Knockdown of circZCCHC2 inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition of TNBC cells in vitro and in vivo. Pirarubicin (THP) treatment downregulated circZCCHC2, and circZCCHC2 affected the sensitivity to THP. CircZCCHC2/miR-1200/translocated promoter region, the nuclear basket protein (TPR) pathway was cascaded and verified. It is demonstrated that circZCCHC2 plays a crucial role in the malignant progression of TNBC via the miR-1200/TPR axis, thereby activating the RAS-RAF-MEK-ERK pathway. The present results indicate that circZCCHC2 has the potential to serve as a novel prognostic biomarker for TNBC.

LncRNA Miat knockdown enhances pirarubicin-mediated anticancer sensitivity in breast cancer cells.

Pirarubicin (THP) is a widely used antitumor agent in clinical practice, but its reduced sensitivity during treatment has limited its use. The aim of this study was to investigate the role and mechanism of LncRNA Miat knockdown in improving THP sensitivity. We assessed the role of Miat overexpression/knockdown on THP-mediated 4T1 anticancer activity by CCK8, TUNEL, flow cytometry, wound healing assay, Transwell, Ca2+ , real time quantitative PCR (RT-qPCR) and Western blot. The results showed that Miat expression was higher in 4T1 mouse breast cancer cells than in HC11 mouse mammary epithelial cells, while THP decreased Miat expression in 4T1. Miat knockdown in combination with further reduced cell viability, promoted apoptosis and inhibited migration compared to THP alone. This may be related to the reduction of calcium ions in 4T1. In conclusion, Miat knockdown enhanced the sensitivity of THP to 4T1 by inhibiting calcium channels.

Astaxanthin protects against pirarubicin-induced H9c2 cardiomyocytes by adjusting microRNA-494-3p-mediated MDM4/p53 signalling pathway.

PURPOSE: Pirarubicin (THP) is an antitumour drug widely used in clinical practice, but its cardiotoxicity limits its application. THP cardiotoxicity must be treated as soon as possible. There is an urgent need to find drugs that alleviate THP cardiotoxicity. The purpose of this study was to investigate the effects and mechanisms of Astaxanthin (AST) on THP-induced cardiomyocytes. METHODS: Rat cardiomyocytes H9c2 were induced with THP. The effects of AST on THP-induced H9c2 and its mechanism were investigated by CCK8, reactive oxygen species assay, tunnel assay, flow cytometry, RT-qPCR, and Western blot. RESULTS: AST increased cell viability, inhibited apoptosis and accelerated cell cycle progression, reduced oxidative damage and inflammatory response in THP-induced H9c2; down-regulated miR-494-3p expression, promoted MDM4 expression, inhibited p53 activation, and suppressed apoptosis-related protein expression. Overexpression of MiR-494-3p reversed the above effects of AST. CONCLUSIONS: AST can inhibit H9c2 apoptosis induced by THP and attenuate H9c2 damage by THP, which may be achieved by downregulating miR-494-3p, upregulating MDM4, and inhibiting p53.

Rapid detection of 10 benzodiazepines and metabolites in blood and urine using DART-MS/MS.

Benzodiazepines are essential screening targets for common sleeping and sedative drugs used in forensic toxicology. Direct analysis in real-time tandem mass spectrometry was used to rapidly identify 10 benzodiazepines and related metabolites in the blood and urine. The related direct analysis in real-time tandem mass spectrometry parameters were optimized. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used for sample preparation. The established method was validated and tested on case specimens. The limits of detection of this method ranged from 0.2 to 20 ng/mL and the limits of quantification from 1 to 50 ng/mL. The recoveries ranged from 78.8% to 114%, and the matrix effects were in the range of -21.2% to 17.9%. The precision and repeatability at high and medium concentrations did not exceed 14.6%, and the limit of quantification did not exceed 18.2%, indicating a desirable linear relationship. The established method was used to determine blood and urine specimens from authentic cases, and promising results were obtained.

LncRNA Miat knockdown protects against pirarubicin-induced cardiotoxicity by targeting miRNA-129-1-3p.

Pirarubicin (THP) is a widely used antitumor drug in clinical practice, but its cardiotoxicity limits its use. The aim of this study was to investigate the protective effect and mechanism of knockdown of lncRNA Miat in THP-induced cardiotoxicity. The extent of damage to immortalized cardiomyocytes in mice was assessed by CCK8, TUNEL, ROS, Ca2+ , RT-qPCR, and Western blot. The relative levels of Miat in THP-treated cardiomyocytes (HL-1) were measured. The protective effect of Miat on THP-treated HL-1 was assessed. The binding relationship between lncRNA Miat and mmu-miRNA-129-1-3p was verified by a dual luciferase reporter gene assay. The protective role of Miat/miRNA-129-1-3p in THP-induced HL-1 was explored by performing a rescue assay. THP reduced cell viability, induced apoptosis, triggered oxidative stress and calcium overload. Expression of Miat in HL-1 was significantly elevated after THP treatment. Miat knockdown significantly alleviated the cardiotoxicity of THP. MiR-129-1-3p is a direct target of Miat. Knockdown of miR-129-1-3p reversed the protective effect of Miat knockdown on HL-1. Miat knockdown can alleviate THP-induced cardiomyocyte injury by regulating miR-129-1-3p.

MiR-494-3p aggravates pirarubicin-induced cardiomyocyte injury by regulating MDM4/p53 signaling pathway.

OBJECTIVE: Pirarubicin (THP) is a widely used antitumor drug in clinical practice, but its cardiotoxicity limits its use. There is an urgent need to find drugs to alleviate the cardiotoxicity of THP. This study aimed to investigate the effect and mechanism of miR-494-3p on THP-induced cardiomyocytes. METHODS: THP induced immortalized mouse cardiomyocytes HL-1, silenced or overexpressed miR-494-3p. The effects of miR-494-3p on HL-1 contained in THP were investigated by CCK8, flow cytometry, ROS detection, JC-1 mitochondrial membrane potential detection, TUNEL cell apoptosis detection, RT-qPCR, and Western blot. RESULTS: miR-494-3p could reduce cell viability, increase oxidative damage, and promote cell apoptosis; at the same time, it inhibited the expression of MDM4, promoted the activation of p53, and promoted the expression of apoptosis-related proteins. MiR-494-3p inhibitors have the opposite effect. CONCLUSION: miR-494-3p can aggravate THP damage to HL-1, which may be achieved by downregulating MDM4 and promoting p53. miR-494-3p is one of the important miRNAs in THP-induced cardiotoxicity, which provides theoretical support for its possible use as a therapeutic target for THP-induced cardiovascular disease.

CircEGFR reduces the sensitivity of pirarubicin and regulates the malignant progression of triple-negative breast cancer via the miR-1299/EGFR axis.

Circular RNAs (circRNAs) have been found to be involved in cancer progression and chemotherapy sensitivity. However, the biological function of circRNAs in triple-negative breast cancer (TNBC) and its effect on the sensitivity to pirarubicin (THP) chemotherapy are still unclear. CircEGFR (hsa_circ_0080220) was screened and verified by bioinformatics analysis, proving it was highly expressed in TNBC cell lines, patient tissues, and plasma exosomes, and was associated with poor prognosis of patients. The expression level of circEGFR in patient tissue has potential diagnostic value to distinguish TNBC tissue from normal breast tissue. In vitro studies confirmed that overexpression of circEGFR promoted the proliferation, migration, invasion, and EMT of TNBC cells and decreased the sensitivity of THP treatment while silencing circEGFR showed the opposite effect. The circEGFR/miR-1299/EGFR pathway was cascaded and verified. CircEGFR regulated malignant progression of TNBC by regulating EGFR via sponging miR-1299. THP can inhibit the malignant phenotype of MDA-MB-231 cells by downregulating the expression of circEGFR. In vivo studies confirmed that overexpression of circEGFR can promote tumor growth and EMT and reduce tumor sensitivity to THP treatment. Silencing circEGFR inhibited the malignant progression of the tumor. These results revealed circEGFR is a promising biomarker for TNBC diagnosis, therapeutic and prognosis.

Direct analysis in real-time tandem mass spectrometry method for the rapid screening of 11 new psychoactive substances in blood and urine.

RATIONALE: With the continuous renewal of new psychoactive substances (NPS), the abuse of NPS has seriously harmed social security and public safety. The number of deaths from the abuse of NPS is increasing year by year. Therefore, there is an immediate need to develop an effective method for detecting NPS. METHODS: Direct analysis in real-time tandem mass spectrometry (DART-MS/MS) was used to detect 11 NPS in blood and urine. The temperature of the ion source was optimized and set to 400°C. The mixture solvent of acetonitrile/methanol (4:1, v/v) was used as the precipitant. SKF-525 (2-(diethylamino)ethyl 2,2-diphenylpentanoate) was selected as the internal standard for quantification. After the pretreatment of the analytes in blood or urine, the supernatant was prepared for instrumental analysis. RESULTS: The results indicated that the correlation coefficients (r2 ) of all analytes ranged from 0.99 to 1 in the linear range. The recoveries of 11 analytes at three spiked levels ranged between 83.4% and 110.4% in blood and between 81.7% and 108.5% in urine. The matrix effects of 11 analytes ranged between 79.5% and 109.5% in blood and between 85.0% and 109.4% in urine. The relative standard deviations of intra-day and inter-day precisions and repeatability were lower than 12.4%, 14.1%, and 14.3% in blood and lower than 11.4%, 13.9%, and 14.3% in urine, respectively. CONCLUSION: The method established for the detection of 11 NPS could meet the needs for the rapid screening of NPS samples. The DART-MS/MS method has the advantages of being efficient, fast, and green. Therefore, it may become a promising technology for the detection of NPS in the future.

Identification of prognostic genes signature and construction of ceRNA network in pirarubicin treatment of triple-negative breast cancer.

BACKGROUND: The altered long non-coding RNA (lncRNA), circular RNA (circRNA) and mRNA expression in triple-negative breast cancer (TNBC) after pirarubicin (THP) treatment can be a critical factor in the development of tumor. Here, we identify a set of lncRNA, circRNA, and mRNA that can reveal the molecular target and molecular mechanism of THP, and can be used to predict the prognostic characteristics of TNBC. METHODS: Affymetrix GeneChip sequencing was performed to determine whether lncRNA, circRNA, and mRNA were changed in MDA-MB-231 cells after THP treatment, and qRT-PCR was used to verify the accuracy of GeneChip results. Bioinformatics methods were used to analyze the differentially expressed (DE) lncRNA, circRNA and mRNA, and the co-expression network and ceRNA network were constructed. The STRING database, Kaplan-meier Mapper database, GEPIA database, and Tumor Immunity Estimation Resource were used to screen hub genes with clinical value and important significance. RESULTS: THP 5 µM could significantly inhibit proliferation, migration and invasion of MDA-MB-231 cells for 24 h. 1547 DE lncRNAs, 4992 DE circRNAs, and 5777 DE mRNAs were identified. The reliability of the GeneChip was verified by qRT-PCR. An mRNA-lncRNA/circRNA co-expression network was constructed based on the Pearson correlation coefficient. Finally, we established a new ceRNA network, including three circRNAs, five miRNAs, and three mRNAs. The mRNAs are associated with immune infiltration. The mRNAs and miRNAs are significantly associated with survival outcomes in TNBC. CONCLUSION: The results reveal the molecular target and mechanism of THP treatment of TNBC. These ceRNA network can be used as molecular targets for the treatment of TNBC patients and as molecular biomarkers to predict patient prognosis.

Icariin alleviates atherosclerosis by regulating the miR-205-5p/ERBB4/AKT signaling pathway.

PURPOSE: Atherosclerosis (AS) is a cardiovascular disease that has become a major threat to public health worldwide. This study aims to elucidate the effect and mechanism of icariin (ICA) in treating atherosclerosis. METHODS: ApoE-/- mouse AS modeling, ELISA, and hematoxylin-eosin staining were conducted to explore whether icariin has a therapeutic effect on AS. The microRNA (miRNA) chips for ICA treatment of ApoE-/- AS mice were developed; in silico analyses were performed, and signaling pathways were identified. Oxidized low-density lipoprotein (Ox-LDL) was used to induce human aortic vascular smooth muscle cells (HAVSMCs) to build an in vitro AS cell model. Moreover, miR-205-5p was silenced. Finally, cell viability was detected by MTT assay, cell apoptosis by flow cytometry and Western blot, and cell migration by the scratch test. RESULTS: ICA could reduce lipid accumulation in the blood vessels of mice and plaque formation to treat AS. ICA promoted apoptosis and inhibited cell migration of HAVSMCs induced by ox-LDL. Moreover, cell proliferation and migration were inhibited via ICA, which was restored by miR-205-5p silencing. CONCLUSION: ICA can alleviate AS and inhibit the proliferation and migration of HAVSMCs induced by ox-LDL, potentially mediated by the upregulation of miR-205-5p.

Identification of a five genes prognosis signature for triple-negative breast cancer using multi-omics methods and bioinformatics analysis.

Triple-negative breast cancer (TNBC) has a high degree of malignancy, lack of effective diagnosis and treatment, and poor prognosis. Bioinformatics methods are used to screen the hub genes and signal pathways involved in the progress of TNBC to provide reliable biomarkers for the diagnosis and treatment of TNBC. Download the raw data of four TNBC-related datasets from the Gene Expression Omnibus (GEO) database and use them for bioinformatics analysis. GEO2R tool was used to analyze and identify differentially expressed (DE) mRNAs. DAVID database was used to carry out gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genome Pathways (KEGG) signal pathway enrichment analysis for DE mRNAs. STRING database and Cytoscape were used to build DE mRNAs protein-protein interaction (PPI) network diagram and visualize PPI network, respectively. Through cytoHubba, cBioPortal database, Kaplan-Meier mapper database, Gene Expression Profiling Interactive Analysis (GEPIA) Database, UALCAN Database, The Cancer Genome Atlas (TCGA) database, Tumor Immunity Estimation Resource identify hub genes. Perform qRT-PCR, Human Protein Atlas analysis, mutation analysis, survival analysis, clinical-pathological characteristics, and infiltrating immune cell analysis. 22 DE mRNAs were identified from the four datasets, including 16 upregulated DE mRNAs and six downregulated DE mRNAs. Enrichment analysis of the KEGG showed that DE mRNAs were principally enriched in pathways in cancer, mismatch repair, cell cycle, platinum drug resistance, breast cancer. Six hub genes were screened based on the PPI network diagram of DE mRNAs. Survival analysis found that TOP2A, CCNA2, PCNA, MSH2, CDK6 are related to the prognosis of TNBC. In addition, mutations, clinical indicators, and immune infiltration analysis show that these five hub genes play an important role in the progress of TNBC and immune monitoring. Compared with MCF-10A, MCF-7, and SKBR-3 cells, TOP2A, PCNA, MSH2, and CDK6 were significantly upregulated in MDA-MB-321 cells. Compared with normal, luminal, and Her-2 positive tissues, CCNA2, MSH2, and CDK6 were significantly upregulated in TNBC. Through comparative analysis of GEO datasets related to colorectal cancer and lung adenocarcinoma, it was determined that these five hub genes were unique differentially expressed genes of TNBC. At last, the hub genes related to the progression, prognosis, and immunity of TNBC have been successfully screened. They are indeed specific to TNBC as prognostic features. They can be used as potential markers for the prognosis of TNBC and provide potential therapeutic targets.

Apocynum venetum leaf extract alleviated doxorubicin-induced cardiotoxicity through the AKT/Bcl-2 signaling pathway.

BACKGROUND: Doxorubicin (DOX) is a broad-spectrum anti-tumor drug that has been associated with cardiotoxicity. Plant extracts have been shown to confer protection against DOX-induced cardiotoxicity. Apocynum venetum L. belongs to the Apocynaceae family. Flavonoid extracted from Apocynum venetum L. possess various biological effects, such as lowering blood pressure levels, sedation, diuresis, anti-aging, and improving immunity. PURPOSE: This study investigated the mechanism by which dry leaf extract of Apocynum venetum L. (AVLE) alleviates DOX-induced cardiomyocyte apoptosis. METHODS: HPLC-MS/MS and HPLC methods were used to analyze the components of AVLE. The effects of DOX and AVLE on apoptosis of H9c2 and HMC cells were assessed using the MTT assay. Calcein AM/PI, TUNEL, and flow cytometry were carried out to determine the effects of AVLE on DOX-induced apoptosis. The effect of AVLE on DOX-induced oxidative stress in cardiomyocytes was investigated using ELISA test. Mito-Tracker Red CMXRos, JC-1, and RT-qPCR assays were performed to evaluate the impact of AVLE on DOX-induced cardiomyocyte mitochondrial activity and membrane permeability. Western blot assay was carried out to determine the activation of multiple signaling molecules, including phosphorylated-protein kinase B (p-AKT), Cytochrome c, Bcl-2 family, and caspase family in the apoptosis pathway. The AKT inhibitor was used to block AKT/Bcl-2 signaling pathway to investigate the role of AKT in the protection conferred by AVLE against DOX-induced cardiotoxicity. RESULTS: A total of 8 compounds, including rutin, hyperoside, isoquercetin, unidentified compounds, myricetin, quercetin, quercetin-3-O-glucuronide and kaempferol, were detected in AVLE. Of note, DOX suppressed lactate dehydrogenase (LDH) levels, aggravated oxidative stress, and promoted cardiomyocyte apoptosis. It also upregulated the mRNA expression levels of voltage-dependent anion channel 1 (VDAC1), adenosine nucleotide transporter 1 (ANT1), and cyclophilin D (CYPD), while suppressing mitochondrial activity and mitochondrial membrane permeability. Treatment with DOX altered the expression levels of apoptosis-associated proteins, Bcl-2 and Bax. However, AVLE treatment alleviated DOX-induced effects on cardiomyocytes. In addition, application of AKT inhibitors promoted DOX-induced apoptosis and reversed the inhibitory effects of AVLE on DOX-induced apoptosis. CONCLUSIONS: AVLE confer cardio protection by suppressing oxidative stress and apoptosis of cardiomyocytes via AKT/Bcl-2 signaling pathway.

Rapid detection of nine synthetic cathinones in blood and urine by direct analysis in real-time-tandem mass spectrometry.

RATIONALE: Designer drugs of cathinone, a kind of hallucinogen, were abused in the recent years. They were also known as bath salts, plant fertilizers, and zombie potions in drug market. The abuse of synthetic cathinones caused many bad effects on social order. Rapid detection of synthetic cathinones became an important subject of study in forensic science. METHODS: Direct analysis in real-time-tandem mass spectrometry (DART-MS/MS) was used to develop an effective method on nine synthetic cathinones in human whole blood and urine. The reference materials with 100 ng/mL were prepared for mass spectrometry optimization with electrospray ionization (ESI) probe tandem QTRAP 4000 mass spectrometer. The temperature of DART ion source was optimized to 400°C. The volumes of 4/1 (v/v) MeCN/MeOH with 0.69 mL were selected for the preparation of 0.31 mL blood and urine samples, respectively. Then the spiked analytes were prepared for detection by the DART 12Dip-it autosampler module. RESULTS: The results showed that the linearities range between 0.1 and 5 µg/mL, the correlation coefficients (r2 ) ranged from 0.99 to 1, the limits of detection (LODs) were all between 0.5 and 50 ng/mL, and the relative standard deviations (RSDs) of repeatability, intra-day and inter-day precisions were all below 13% and 14% in blood and urine, respectively. CONCLUSION: The results indicated that the method could meet the needs of rapid screening of samples that may contain synthetic cathinones. In addition, the method developed has many advantages, such as efficient, fast sample preparation, and environmental protection. Therefore, the DART-MS/MS method would provide effective data support for rapid screening of synthetic cathinones in forensic science.