Xiaojian Zhang

Background: RNA 5-methylcytosine (m⁵C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m⁵C modification in tumor drug resistance remain unclear. Methods: The correlation between RNA m⁵C methylation, m⁵C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m⁵C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. Results: RNA m⁵C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m⁵C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m⁵C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m⁵C reader Y-box binding protein 1 (YBX1). Conclusions: Our study reveals a critical function of aberrant RNA m⁵C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

Background: Ticagrelor and clopidogrel are commonly used antiplatelet agents, and we conducted a pharmacovigilance analysis using the Food and Drug Administration Adverse Event Reporting System (FAERS) to provide a reference for safe and reasonable clinical use.Methods: Data were collected in FAERS from 2012 Q1 to 2022 Q2 for data cleaning. We used system organ classes (SOCs) and prefer terms (PTs) from the Medical Dictionary of Regulatory Activity (MedDRA version 25.1). Adverse event reports were retrieved at the PT level. Adverse reaction (ADR) signals of ticagrelor and clopidogrel were mined by calculating reporting odds ratios (ROR), proportional reporting ratios (PRR), information component (IC) and empirical Bayesian geometric mean (EBGM). After that, further analysis of the hemorrhagic signals and their clinical information were performed.Results: The number of ADR reports where the primary suspect (PS) drugs were 15,133 for ticagrelor and 23,860 for clopidogrel. Significant ADR signals were identified by the SOC analysis for ticagrelor including cardiac disorders (ROR 4.87, PRR 4.46), respiratory disorders (ROR 2.45, PRR 2.28), and vascular disorders (ROR 2.22, PRR 2.16). Clopidogrel included blood disorders (ROR 2.86, PRR 2.77), vascular disorders (ROR 2.71, PRR 2.61), and cardiac disorders (ROR 2.29, PRR 2.22). At the PT level, the more frequent ADR signals for ticagrelor were dyspnoea, contusion, and haemorrhage, while clopidogrel were gastrointestinal haemorrhage, anaemia, and drug interaction. The hemorrhagic signals of both were mainly focused on the SOC level of gastrointestinal disorders, injury disorders and vascular disorders and nervous system disorders. The death and life-threatening rate of ticagrelor was 7.76 percentage higher than that of clopidogrel.Conclusion: Clinicians need to pay attention to not only common ADRs but also be alert to new ADR signals when choosing to use ticagrelor and clopidogrel. This study provides a reference for the reasonable and safe clinical use of ticagrelor and clopidogrel.

Fraxetin, a natural compound present in many dietary supplements and herbs, is useful in the treatment of acute bacillary dysentery and type 2 diabetes. Previously, several metabolic studies have revealed extensive first-pass metabolism causing formation of fraxetin-O-glucuronides (G1 and G2), resulting in poor bioavailability of fraxetin. Active transport processes play an important role in the excretion of fraxetin-O-glucuronides. Nevertheless, the transporters involved are yet to be elucidated. In this study, we aimed to determine the active efflux transporters, including breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs), involved in the excretion of fraxetin-O-glucuronides. A chemical inhibitor, MK571 (5 and 20 μM), a pan-MRP inhibitor, led to a significant decrease in excreted G1 (maximal 59.1%) and G2 levels (maximal 42.4%), whereas Ko143 (5 and 20 μM), a selective BCRP inhibitor, caused moderate downregulation of excreted G1 (maximal 29.4%) and G2 (maximal 28.5%). Furthermore, MRP3 silencing resulted in a marked decrease of excretion rates (by 29.1% for G1 and by 21.1% for G2) and of fraction metabolized (f_met; by 24.1% for G1 and by 18.6% for G2). Similar results, i.e., a significant reduction in excretion rates (by 34.8% for G1 and by 32.3% for G2) and in f_met (by 22.7% for G1 and by 23.1% for G2) were obtained when MRP4 was partially silenced. No obvious modifications in the excretion rates, intracellular levels, and f_met values of glucuronides were observed after short hairpin RNA (shRNA)-mediated silencing of transporters BCRP and MRP1. Taken together, our results indicate that MRP3 and MRP4 contribute more to the excretion of fraxetin-O-glucuronides than the other transporters do.

Previous studies have demonstrated that miRNAs play an important role in tumor development and progression. The role of miR-320d has been studied in several cancers except for glioma. The aim of the study was to investigate the expression levels, biological function, and mechanism of miR-320d in glioma. The expression levels of miR-320d were detected in glioma tissues and cell lines (U87 and U251) by RT-qPCR. Cell proliferation, colony formation, apoptosis, cell cycle and transwell assays were performed in glioma cell lines transfected with miR-320d mimics or controls to evaluate the effects of miR-320d in vitro. The expression levels of invasive-related proteins were determined by western blot analysis. Results showed that the expression of miR-320d was significantly decreased in glioma tissues and cell lines. Overexpression of miR-320d could significantly suppress cell growth, migration and invasion, and induced cell apoptosis as well as cell cycle at G0/G1 arrest in U87 and U251 cell lines. Additionally, expression levels of MMP-2, MMP-9, N-cadherin and Integrin-β1 reduced, while E-cadherin increased in miR-320d mimic group. Overall, the present study is the first to demonstrate that miR-320d may serve as an independent prognostic factor, indicating that miR-320d is a biomarker for prognosis and therapy in glioma.

Aims It has been proven that carbon nanoparticles or diesel exhaust particles stimulate platelet activation. However, the effect of fine particle matter (PM_2.5) on platelet activation remains unknown, which motivates this study. Methods PM_2.5 samples were collected in an urban area of Zhengzhou, China. To study the morphological characteristics and the mass concentrations of trace elements of PM_2.5 samples, a filed-emission scanning electron microscope, the Image-J software, and an inductively coupled plasma mass spectrometry were used. Washed human platelets or platelet-rich-plasma were used to study the effect of PM_2.5 on platelet aggregation, P-selectin expression, or platelet signaling pathways. The cytotoxicity in platelets exposed to PM_2.5 was evaluated by a lactate dehydrogenase assay kit. In addition, platelet adhesion and spreading were studied on collagen-coated surfaces in stable conditions. Results The filed-emission scanning electron microscope scanning showed that PM_2.5 samples varied in shape and size distributions. The mean equivalent spherical diameter of these particles was 1.97 ± 0.04 μm, of which 82.40% were particles with equivalent spherical diameters of less than 2.5 μm. The mass concentration of Ca was higher than that of other elements. The other elements followed the trend of Al>Fe>Zn>Mg>Pb>K>Mn>Cu>Ti>Ba>As>Sr>Sn>Sb>Cd>B>Se>Mo>Ag>Ni>TI>V>Co. Furthermore, pretreatment of PM_2.5 significantly inhibited rather than potentiated collagen-induced platelet aggregation and P-selectin expression, whereas it had no significant effect on ADP-induced platelet aggregation and P-selectin expression. The lactate dehydrogenase analysis showed trivial cytotoxic effect of PM_2.5 exposure on platelets. Pretreatment of PM_2.5 inhibited platelet adhesion on immobilized collagen-coated surfaces; however, it almost did not impact the platelet spreading. Immunoblotting analysis indicated that PM_2.5 reduced collagen-induced phosphorylation of phospholipase C gamma-2 (PLCγ2) at Tyr759, Akt at Ser473, and glycogen synthase kinase 3β (GSK3β) at Ser9. Conclusions PM_2.5 attenuated collagen-induced platelet aggregation, α-granule secretion and adhesion, with the potential mechanism of impairing PLCγ2, Akt, and GSK3β signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 530–540, 2017.