Variant rs8400 enhances ALKBH5 expression through disrupting miR-186 binding and promotes neuroblastoma progression

Objective:AlkB homolog 5(ALKBH5)has been proven to be closely related to tumors.However,the role and molecular mechanism of ALKBH5 in neuroblastomas have rarely been reported.Methods:The potential functional single-nucleotide polymorphisms(SNPs)in ALKBH5 were identified by National Center for Biotechnology Information(NCBI)dbSNP screening and SNPinfo software.TaqMan probes were used for genotyping.A multiple logistic regression model was used to evaluate the effects of different SNP loci on the risk of neuroblastoma.The expression of ALKBH5 in neuroblastoma was evaluated by Western blotting and immunohistochemistry(IHC).Cell counting kit-8(CCK-8),plate colony formation and 5-ethynyl-2'-deoxyuridine(EdU)incorporation assays were used to evaluate cell proliferation.Wound healing and Transwell assays were used to compare cell migration and invasion.Thermodynamic modelling was performed to predict the ability of miRNAs to bind to ALKBH5 with the rs8400 G/A polymorphism.RNA sequencing,N6-methyladenosine(mA)sequencing,mA methylated RNA immunoprecipitation(MeRIP)and a luciferase assay were used to identify the targeting effect of ALKBH5 on SPP1.Results:ALKBH5 was highly expressed in neuroblastoma.Knocking down ALKBH5 inhibited the proliferation,migration and invasion of cancer cells.miR-186-3p negatively regulates the expression of ALKBH5,and this ability is affected by the rs8400 polymorphism.When the G nucleotide was mutated to A,the ability of miR-186-3p to bind to the 3'-UTR of ALKBH5 decreased,resulting in upregulation of ALKBH5.SPPI is the downstream target gene of the ALKBH5 oncogene.Knocking down SPP1 partially restored the inhibitory effect of ALKBH5 downregulation on neuroblastoma.Downregulation of ALKBH5 can improve the therapeutic efficacy of carboplatin and etoposide in neuroblastoma.Conclusions:We first found that the rs8400 G>A polymorphism in the m6A demethylase-encoding gene ALKBH5 increases neuroblastoma susceptibility and determines the related mechanisms.The aberrant regulation of ALKBH5 by miR-186-3p caused by this genetic variation in ALKBH5 promotes the occurrence and development of neuroblastoma through the ALKBH5-SPP1 axis.

基于血清药物化学和网络药理学的荜茇抗胃溃疡药效物质基础分析

荜茇(PL)是具有药食两用性的常用中药,中医临床多生用,具有温中散寒、行气止痛的功效,用于胃脘冷痛、胃寒呕吐等,同时也是蒙医、藏医和印度传统常用药物。在本研究中,首先建立大鼠胃溃疡动物模型,考察荜茇对胃粘膜的保护作用,进而将血清药物化学与网络药理学相结合,挖掘荜茇药效物质基础,从荜茇中筛选药效物质和潜在作用靶点。研究结果表明,荜茇对乙醇诱导的胃溃疡具有显著的保护作用;网络药理学预测了潜在的药物-疾病共同靶点后,GO富集分析预测荜茇可能影响多种生物过程,如脂质代谢和炎症反应。KEGG富集分析提示TNF和AGE-RAGE信号通路可能参与其中,分子对接预测芝麻素与胡椒碱等可能是荜茇抗炎和抗菌作用的主要成分。此外,荜茇的血清药物化学揭示了入血成分中有23种原型成分和17种代谢物。本研究整合了血清药物化学和网络药理学,为荜茇在胃溃疡治疗中的药效物质研究提供了实验依据。

PTBP2-Mediated Alternative Splicing of IRF9 Controls Tumor-Associated Monocyte/Macrophage Chemotaxis and Repolarization in Neuroblastoma Progression

The recurrence and metastasis of children with mediastinal neuroblastoma(NB)are also occurred after surgery,chemotherapy,or radiotherapy.Strategies targeting the tumor microenvironment have been reported to improve survival;however,thorough investigations of monocytes and tumor-associated macrophages(Mϕs)with specialized functions in NB are still lacking.Our data first demonstrated polypyrimidine tract binding protein 2(PTBP2)as a possible identifier in patients with mediastinal NB screened by proteomic profiling and that PTBP2 predicted good outcomes.Functional studies revealed that PTBP2 in NB cells induced the chemotactic activity and repolarization of tumor-associated monocytes and Mϕs,which,in turn,inhibited NB growth and dissemination.Mechanistically,PTBP2 prevents interferon regulatory factor 9 alternative splicing and upregulates signal transducers and activators of transcription 1 to stimulate C-C motif chemokine ligand 5(CCL5)and interferon-stimulated gene factor-dependent type I interferon secretion,to induce monocyte/Mϕs chemotaxis,and to sustain monocytes in a proinflammatory phenotype.Our study defined a critical event of PTBP2-induced monocytes/Mϕs in NB progression and revealed that RNA splicing occurred by PTBP2 benefits immune compartmentalization between NB cells and monocytes.This work revealed the pathological and biological role of PTBP2 in NB development and indicates that PTBP2-induced RNA splicing benefits immune compartmentalization and predicted a favorable prognosis in mediastinal NB.

UPLC-Q-Exactive-MS同时测定三臣丸中六种活性成分的药代动力学研究（英文）

建立三臣丸药代动力学的UPLC-Q-Exactive-MS检测方法,同时测定大鼠血浆中胆酸、猪胆酸、羟基红花黄色素A、山奈酚-3-O-芸香糖苷、脱水红花黄色素B和紫丁香苷6种活性成分的含量。HPLC分离采用SHIMADZU GIST C18色谱柱,流动相为乙腈–0.1%甲酸溶液梯度洗脱。质谱检测采用HESI离子源负离子检测模式。6种活性成分的线性范围0.50–1280 ng/m L,r均大于0.99;日内及日间精密度(RSD)均小于12.7%;提取回收率、基质效应和稳定性的计算值均符合FDA生物分析方法验证准则。该文中的三臣丸为传统的蒙药复方,由牛黄、红花和天竺黄三味药材等量组成。该方法为首次报道,并且已成功应用于三臣丸的药代动力学研究。

Liver DNA methylation and transcriptome between 1- and 3-year-old grass carp

Diseases of grass carps often occur in juveniles but not in adults that may have established disease resistance during development.We performed both DNA bisulfite and transcriptome sequencing on liver libraries of 1-and 3-year-old grass carps.Differential DNA-methylation analysis exhibited a declined methylation level through development.Functional annotation revealed that identified differentially methylated genes(DMGs)and differentially expressed genes were enriched in immune-related pathways such as PI3K-Akt signaling pathway and its related pathways.Both differentially methylated and differentially expressed genes were clustered into the growth-and immune-related function networks.Subcellular localization analysis indicated that the DMGs localized on cell membrane were significantly enriched in calcium signaling pathway and neuroactive ligand-receptor interaction pathway,implying the importance of G protein-coupled receptors to development.These findings will broaden our understanding of the key genes and pathways that affect the immune system at different development stages and the developing protective strategies in grass carp.