Identification of Metabolite Interference Is Necessary for Accurate LC-MS Targeted Metabolomics Analysis.

Targeted metabolomics has been broadly used for metabolite measurement due to its good quantitative linearity and simple metabolite annotation workflow. However, metabolite interference, the phenomenon where one metabolite generates a peak in another metabolite's MRM setting (Q1/Q3) with a close retention time (RT), may lead to inaccurate metabolite annotation and quantification. Besides isomeric metabolites having the same precursor and product ions that may interfere with each other, we found other metabolite interferences as the result of inadequate mass resolution of triple-quadruple mass spectrometry and in-source fragmentation of metabolite ions. Characterizing the targeted metabolomics data using 334 metabolite standards revealed that about 75% of the metabolites generated measurable signals in at least one other metabolite's MRM setting. Different chromatography techniques can resolve 65-85% of these interfering signals among standards. Metabolite interference analysis combined with the manual inspection of cell lysate and serum data suggested that about 10% out of ∼180 annotated metabolites were mis-annotated or mis-quantified. These results highlight that a thorough investigation of metabolite interference is necessary for accurate metabolite measurement in targeted metabolomics.

Quantitative analysis of a novel DNA hypermethylation panel using bronchial specimen for lung cancer diagnosis.

Non-diagnostic findings are common in transbronchial lung biopsy (TBLB) and endobronchial ultrasound-guided transbronchial lung biopsy (EBUS-TBLB). One of the challenges is to improve the detection of lung cancer using these techniques. To address this issue, we utilized an 850 K methylation chip to identify methylation sites that distinguish malignant from benign lung nodules. Our study found that a combination of HOXA7, SHOX2 and SCT methylation analysis has the best diagnostic yield in bronchial washing (sensitivity: 74.1%; AUC: 0.851) and brushing samples (sensitivity: 86.1%; AUC: 0.915). We developed a kit comprising these three genes and validated it in 329 unique bronchial washing samples, 397 unique brushing samples and 179 unique patients with both washing and brushing samples. The panel's accuracy in lung cancer diagnosis was 86.9%, 91.2% and 95% in bronchial washing, brushing and washing + brushing samples, respectively. When combined with cytology, rapid on-site evaluation (ROSE), and histology, the panel's sensitivity in lung cancer diagnosis was 90.8% and 95.8% in bronchial washing and brushing samples, respectively, and 100% in washing + brushing samples. Our findings suggest that quantitative analysis of the three-gene panel can improve the diagnosis of lung cancer using bronchoscopy.

A DNA methylation signature for the prediction of tumour recurrence in stage II colorectal cancer.

BACKGROUND: A major challenge in stage II colorectal carcinoma is to identify patients with increased risk of recurrence. Biomarkers that distinguish patients with poor prognosis from patients without recurrence are currently lacking. This study aims to develop a robust DNA methylation classifier that allows the prediction of recurrence and chemotherapy benefit in patients with stage II colorectal cancer. We performed a genome-wide DNA methylation capture sequencing in 243 stage II colorectal carcinoma samples and identified a relapse-specific DNA methylation signature consisting of eight CpG sites. METHODS: Two hundred and forty-three patients with stage II CRC were enrolled in this study. In order to select differential methylation sites among recurrence and non-recurrence stage II CRC samples, DNA methylation profiles of 62 tumour samples including 31 recurrence and 31 nonrecurrence samples were analysed using the Agilent SureSelectXT Human Methyl-Seq, a comprehensive target enrichment system to analyse CpG methylation. Pyrosequencing was applied to quantify the methylation level of candidate DNA methylation sites in 243 patients. Least absolute shrinkage and selection operator (LASSO) method was employed to build the disease recurrence prediction classifier. RESULTS: We identified a relapse-related DNA methylation signature consisting of eight CpG sites in stage II CRC by DNA methylation capture sequencing. The classifier showed significantly higher prognostic accuracy than any clinicopathological risk factors. The Kaplan-Meier survival curve showed an association of high-risk score with poor prognosis. In multivariate analysis, the signature was the most significant prognosis factor, with an HR of 2.80 (95% CI, 1.71-4.58, P < 0.001). The signature could identify patients who are suitable candidates for adjuvant chemotherapy. CONCLUSIONS: An eight-CpG DNA methylation signature is a reliable prognostic and predictive tool for disease recurrence in patients with stage II CRC.

Customized Deep Eutectic Solvents as Green Extractants for Ultrasonic-Assisted Enhanced Extraction of Phenolic Antioxidants from Dogbane Leaf-Tea.

This study evaluates the application of eco-friendly deep eutectic solvents (DESs) in the extraction of phenolic antioxidants from dogbane leaf-tea (DLT). The results showed DESs with lower viscosity allowed an efficient extraction of significantly higher contents of total phenolics or flavonoids. An innovative and high-efficient solvent, choline chloride-levulinic acid (ChCl-LevA), was screened and used in ultrasonic-assisted extraction (UAE) of phenolic compounds from DLT. According to full factorial design experimental results, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and anti-α-glucosidase activity (α-GIA) of the DLT extracts were simultaneously optimized by response surface methodology. Sonication temperature and water content in ChCl-LevA were found to be the major factors affecting the TPC, TFC, antioxidant activity, and α-GIA of DLT extracts. Under the optimum parameters (water content in ChCl-LevA was 45%, sonication temperature was 50 °C, and extraction time was 30 min), the measured results for all the responses were obtained as follows: TPC-91.38 ± 7.20 mg GAE/g DW, TFC-84.12 ± 3.47 mg RE/g DW, ABTS+-492 ± 7.33 mmol TE/g DW, FRAP-6235 ± 121 µmol Fe(II)/g DW and α-GIA-230 ± 7.59 mmol AE/g DW, which were consistent with the predicted values. In addition, strongly significant positive correlations were observed between TPC/TFC and bio-activities of the DLT extracts. HPLC results indicated high contents of (-)-epigallocatechin (4272 ± 84.86 µg/g DW), catechin (5268 ± 24.53 µg/g DW), isoquercitrin (3500 ± 86.07 µg/g DW), kaempferol 3-O-rutinoside (3717 ± 97.71 µg/g DW), and protocatechuic acid (644 ± 1.65 µg/g DW) were observed in the DLT extracts. In contrast to other extraction methods, ChCl-LevA-based UAE yielded higher TPC, TFC, individual phenolic contents, stronger antioxidant activity, and α-GIA. Scanning electron microscope (SEM) analysis further confirmed that ChCl-LevA-based UAE enhanced the disruption of cell wall structure, thereby making more phenolic antioxidants released from DLT. In short, ChCl-LevA-based UAE was confirmed to be an innovative and high-efficient method for extraction of phenolic antioxidants from DLT. Dogbane leaves can be considered as a good tea source rich in natural antioxidants.

LC-Q-Orbitrap-MS/MS Characterization, Antioxidant Activity, and α-Glucosidase-Inhibiting Activity With In Silico Analysis of Extract From Clausena Indica (Datz.) Oliv Fruit Pericarps.

Clausena indica (Datz.) Oliv fruit pericarps (CIOPs) is an important agro-industrial by-product rich in active components. In this article, the effects of traditional and green deep eutectic solvents (DESs) on the high-performance liquid chromatography (HPLC) characterization, antioxidant activities, and α-glucosidase-inhibitory activity of phenolic extracts from CIOPs were investigated for the first time. The results showed that ChCl-Gly and Bet-CA had higher extraction efficiency for the total phenolic content (TPC, 64.14-64.83 mg GAE/g DW) and total flavonoid content (TFC, 47.83-48.11 mg RE/g DW) compared with the traditional solvents (water, methanol, and ethyl acetate). LC-Q-Orbitrap-MS/MS was adopted to identify the phenolic compositions of the CIOPs extracts. HPLC-diode array detection (HPLC-DAD) results indicated that arbutin, (-)-epigallocatechin, chlorogenic acid, procyanidin B1, (+)-catechin, and (-)-epicatechin were the major components for all extracts, especially for deep eutectic solvents (DESs). In addition, ChCl-Xyl and ChCl-Gly extracts showed higher antioxidant activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS+•), ferric reducing antioxidant power (FRAP), reducing power (RP), and cupric ion reducing antioxidant capacity (CUPRAC) than extracts extracted by other solvents. A strong α-glucosidase-inhibiting activity (IC50, 156.25-291.11 µg/ml) was found in three DESs extracts. Furthermore, in silico analysis of the major phenolics in the CIOPs extracts was carried out to explore their interactions with α-glucosidase. Multivariate analysis was carried out to determine the key factors affecting the antioxidant activity and α-glucosidase-inhibiting activity. In short, DES can be taken as a promising solvent for valorization and recovery of bioactive compounds from agro-industrial by-products. The results verified that CIOPs can be used as a prospective source rich in bio-active compounds applied in the food and pharmacy industries.

Isolation of Protein Complexes Associated with Long Non-coding RNAs.

Long non-coding RNAs (lncRNAs) are a new class of regulatory genes that play critical roles in various processes ranging from normal development to human diseases. Recent studies have shown that protein complexes are required for the functions of lncRNAs. The identification of these proteins which are associated with lncRNAs is critical for the understanding of molecular mechanisms of lncRNAs in gene regulation and their functions. In this chapter, we describe a method to isolate proteins associated with lncRNAs. This procedure involves fusion protein Maltose-Binding Protein (MBP) fused to MS2-binding protein to pull down the proteins associated with lncRNA and the identification of these proteins by mass spectrometry.

ZBTB7A, a miR-663a target gene, protects osteosarcoma from endoplasmic reticulum stress-induced apoptosis by suppressing LncRNA GAS5 expression.

Many studies have uncovered the essential role of ZBTB7A in regulating tumourigenesis. However, its functional significance in cell responses to endoplasmic reticulum stress (ER stress) remains poorly understood. Here we report that ZBTB7A functions as an important prosurvival factor in osteosarcoma cells undergoing pharmacological ER stress-induced by tunicamycin (TM) or thapsigargin (TG). The downregulation of ZBTB7A expression by ER stress promoted cell apoptosis in vitro and in vivo. ZBTB7A expression levels were increased in osteosarcoma tissues and elevated ZBTB7A was associated with osteosarcoma metastasis. Further mechanistic studies revealed that miR-663a induced by ER stress directly bound to the 3'UTR of ZBTB7A and contributed to ER stress-induced ZBTB7A downregulation in osteosarcoma cells. Additionally, our data revealed that ZBTB7A bound to the promoter of LncRNA GAS5 and transcriptionally suppressed LncRNA GAS5 expression, leading to a decline in ER stress-induced cell apoptosis. Collectively, our findings reveal the prosurvival role of ZBTB7A in osteosarcoma adaptation to ER stress and suggest that the miR-663a-ZBTB7A-LncRNAGAS5 pathway is essential for the survival of human osteosarcoma cells under ER stress.

Reciprocal regulation of integrin ß4 and KLF4 promotes gliomagenesis through maintaining cancer stem cell traits.

BACKGROUND: The dismal prognosis of patients with glioma is largely attributed to cancer stem cells that display pivotal roles in tumour initiation, progression, metastasis, resistance to therapy, and relapse. Therefore, understanding how these populations of cells maintain their stem-like properties is critical in developing effective glioma therapeutics. METHODS: RNA sequencing analysis was used to identify genes potentially involved in regulating glioma stem cells (GSCs). Integrin ß4 (ITGB4) expression was validated by quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) staining. The role of ITGB4 was investigated by flow cytometry, mammosphere formation, transwell, colony formation, and in vivo tumorigenesis assays. The reciprocal regulation between Integrin ß4 and KLF4 was investigated by chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, immunoprecipitation, and in vivo ubiquitylation assays. RESULTS: In this study, we found that ITGB4 expression was increased in GSCs and human glioma tissues. Upregulation of ITGB4 was correlated with glioma grades. Inhibition of ITGB4 in glioma cells decreased the self-renewal abilities of GSCs and suppressed the malignant behaviours of glioma cells in vitro and in vivo. Further mechanistic studies revealed that KLF4, an important transcription factor, directly binds to the promoter of ITGB4, facilitating its transcription and contributing to increased ITGB4 expression in glioma. Interestingly, this increased expression enabled ITGB4 to bind KLF4, thus attenuating its interaction with its E3 ligase, the von Hippel-Lindau (VHL) protein, which subsequently decreases KLF4 ubiquitination and leads to its accumulation. CONCLUSIONS: Collectively, our data indicate the existence of a positive feedback loop between KLF4 and ITGB4 that promotes GSC self-renewal and gliomagenesis, suggesting that ITGB4 may be a valuable therapeutic target for glioma.

KLF4, a miR-32-5p targeted gene, promotes cisplatin-induced apoptosis by upregulating BIK expression in prostate cancer.

BACKGROUND: Chemotherapeutic insensitivity remains a big challenge in prostate cancer treatment. Recently, increasing evidence has indicated that KLF4 plays a key role in prostate cancer. However, the potential biological role of KLF4 in Chemotherapeutic insensitivity of prostate cancer is still unknown. METHODS: The role of KLF4 in cisplatin-induced apoptosis was detected by western blotting and a cell counting kit (CCK8). The potential molecular mechanism of KLF4 in regulating prostate cancer chemosensitivity was investigated by RNA sequencing analysis, q-RT-PCR, western blotting and chromatin immunoprecipitation (ChIP). The expression level of KLF4 mediated by miR-32-5p was confirmed by bioinformatic analysis and luciferase assays. RESULTS: Here, we found that KLF4 was induced by cisplatin in prostate cancer cells and that the increase in KLF4 promoted cell apoptosis. Further mechanistic studies revealed that KLF4 directly bound to the promoter of BIK, facilitating its transcription. Additionally, we also found that the gene encoding KLF4 was a direct target of miR-32-5p. The downregulation of miR-32-5p in response to cisplatin treatment promoted KLF4 expression, which resulted in a increase in the chemosensitivity of prostate cancer. CONCLUSION: Thus, our data revealed that KLF4 is an essential regulator in cisplatin-induced apoptosis, and the miR-32-5p-KLF4-BIK signalling axis plays an important role in prostate cancer chemosensitivity.

Regulation of the adaptation to ER stress by KLF4 facilitates melanoma cell metastasis via upregulating NUCB2 expression.

BACKGROUND: Adaptation to ER stress has been indicated to play an important role in resistance to therapy in human melanoma. However, the relationship between adaptation to ER stress and cell metastasis in human melanoma remains unclear. METHODS: The relationship of adaptation to ER stress and cell metastasis was investigated using transwell and mouse metastasis assays. The potential molecular mechanism of KLF4 in regulating the adaptation to ER stress and cell metastasis was investigated using RNA sequencing analysis, q-RT-PCR and western blot assays. The transcriptional regulation of nucleobindin 2 (NUCB2) by KLF4 was identified using bioinformatic analysis, luciferase assay, and chromatin immunoprecipitation (ChIP). The clinical significance of KLF4 and NUCB2 was based on human tissue microarray (TMA) analysis. RESULTS: Here, we demonstrated that KLF4 was induced by ER stress in melanoma cells, and increased KLF4 inhibited cell apoptosis and promoted cell metastasis. Further mechanistic studies revealed that KLF4 directly bound to the promoter of NUCB2, facilitating its transcription. Additionally, an increase in KLF4 promoted melanoma ER stress resistance, tumour growth and cell metastasis by regulating NCUB2 expression in vitro and in vivo. Elevated KLF4 was found in human melanoma tissues, which was associated with NUCB2 expression. CONCLUSION: Our data revealed that the promotion of ER stress resistance via the KLF4-NUCB2 axis is essential for melanoma cell metastasis, and KLF4 may be a promising specific target for melanoma therapy.

Integrin-ß5, a miR-185-targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating ß-catenin stability.

BACKGROUND: The tumour microenvironment is essential for cancer progress and metastasis. Integrin-ß5 (ITGB5), a member of the integrin family, has been implicated to mediate the interactions of cells with the extracellular matrix (ECM) and promote tumorigenesis in several malignancies. However, the role of ITGB5 in hepatocellular carcinoma (HCC) is still unknown. METHODS: The biological function of ITGB5 in HCC was investigated using migration, colony formation assays. The potential molecular mechanism of ITGB5 in regulating HCC tumorigenesis and ß-catenin stabilization was investigated by western blotting, co-immunoprecipitation and ubiquitination assays. The expression level of ITGB5 mediated by miR-185 was confirmed by bioinformatic analysis, luciferase assay. The clinical significance of ITGB5 was based on human tissue microarray (TMA) analysis. RESULTS: Here, we found that the expression of ITGB5 is increased in HCC tissues. Elevated ITGB5 markedly facilitates HCC cell migration and tumorigenesis in vitro and in vivo. Further mechanistic studies revealed that ITGB5, as a partner of ß-catenin, directly interacts with ß-catenin and inhibits its degradation, thus leading to WNT/ß-catenin activity. Subsequently, we also found that ITGB5 is a direct targeted gene of miR-185. The downregulation of miR-185 in HCC cells promotes an increase in ITGB5. An additional increase of ITGB5 is associated with ß-catenin upregulation and a miR-185 decrease in HCC tissues. CONCLUSIONS: Our data reveal that the miR-185-ITGB5-ß-catenin pathway plays an important role in HCC tumorigenesis, and ITGB5 may be a promising specific target for HCC therapy.

ZBTB7A Enhances Osteosarcoma Chemoresistance by Transcriptionally Repressing lncRNALINC00473-IL24 Activity.

Chemoresistance remains a major drawback to osteosarcoma treatment. ZBTB7A, a member of the POK transcription repressor family, was shown to play an important role in tumorigenesis. However, the effect of ZBTB7A on osteosarcoma chemoresistance is completely unknown. In this study, we found that ZBTB7A is increased in cisplatin-resistant osteosarcoma cells and that elevated ZBTB7A inhibits cisplatin-induced apoptosis by repressing LINC00473 expression. Further mechanistic studies revealed that ZBTB7A directly binds to the promoter and suppresses the transcription of LINC00473. Additionally, our data indicate that LINC00473 interacts with the transcript factor C/EBPß, facilitating its binding to the promoter of IL24, leading to decrease chemoresistance. Thus, these findings indicate that the ZBTB7A-mediated LINC00473-C/EBPß-IL24 pathway is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

LncRNAAC132217.4, a KLF8-regulated long non-coding RNA, facilitates oral squamous cell carcinoma metastasis by upregulating IGF2 expression.

Increasing evidence has revealed the aberrant expression of long non-coding RNAs (lncRNAs) in many cancer types, including oral squamous cell carcinoma (OSCC). However, limited investigations report metastasis-related lncRNAs in OSCC. Herein, we report the identification of dysregulated intergenic lncRNAs in the highly metastatic OSCC cell line, UM-SCC6H. One of the lncRNAs, termed AC132217.4, was remarkably upregulated and promoted cell migration and epithelial-mesenchymal transition (EMT) by upregulating IGF2 expression. Further mechanistic studies revealed that AC132217.4 interacted with the 3'UTR of IGF2 mRNA and increased its stability, leading to increased IGF2 levels. Thereafter, we found that KLF8 binds to the upstream sequence of AC132217.4, activating its expression at the transcriptional level, which accelerated OSCC metastasis via the AC132217.4-IGF2 axis both in vitro and in vivo. We also revealed that the expression level of AC132217.4 was increased in OSCC tissues, and this elevation correlated with KLF8 and IGF2 expression. Thus, our data demonstrate that the KLF8-AC132217.4-IGF2 signalling pathway plays a critical role in OSCC metastasis.

Identification and Functional Divergence Analysis of WOX Gene Family in Paper Mulberry.

The WOX (WUSCHEL-related homeobox) is a plant-specific transcription factor involved in plant development and stress response. However, few studies have been reported on the WOX gene in woody plants. In this study, 10 BpWOX genes were isolated from paper mulberry by RACE-PCR and categorized into three clades through phylogenetic analysis, ancient, intermediate and WUS clade. Among them, five members had the transcriptional activity detected by yeast one-hybrid and seven were uniquely localized to the nucleus through green fluorescent protein (GFP) observation. The expression patterns of BpWOX genes in different tissues and under diverse treatments were quantified by the qRT-PCR method. Results showed that BpWUS was expressed in the apical bud, stem and root, BpWOX5 and BpWOX7 functioned only in the root tip, and three BpWOXs regulated leaf development redundantly. BpWOX9 and BpWOX10 were induced by indole-3-acetic acid (IAA) or jasmonic acid (JA), while BpWOX2 was repressed by five phytohormones. Interestingly, most BpWOX genes were responsive to the abiotic stress stimuli of drought, salt, cold, and cadmium (CdCl2). Together, our study revealed that BpWOXs were functionally divergent during paper mulberry development and environmental adaptation, which might be related to their evolutionary relationships. Our work will benefit the systematic understanding of the precise function of WOX in plant development and environmental stress responses.