目的:探讨乳酸对三阴性乳腺癌(triple-negative breast cancer,TNBC)细胞恶性行为的影响,并揭示胰岛素样生长因子2 mRNA结合蛋白3(insulin-like growth factor 2 m RNA binding protein 3,IGF2BP3)通过m6A依赖性方式调控EP300,进而介导...目的:探讨乳酸对三阴性乳腺癌(triple-negative breast cancer,TNBC)细胞恶性行为的影响,并揭示胰岛素样生长因子2 mRNA结合蛋白3(insulin-like growth factor 2 m RNA binding protein 3,IGF2BP3)通过m6A依赖性方式调控EP300,进而介导代谢重编程的表观遗传学机制。方法:通过外源性添加乳酸钠处理TNBC细胞,采用CCK-8、集落形成、划痕愈合和Transwell实验检测细胞增殖与迁移能力;利用shRNA慢病毒载体构建IGF2BP3稳定敲低细胞模型,结合Western blot分析组蛋白乳酸化修饰水平;进一步通过RIP-seq、MeRIP-seq筛选IGF2BP3的m6A靶基因,并利用qRT-PCR和Western blot验证关键分子表达。结果:乳酸钠处理显著增强了TNBC细胞的增殖和迁移能力,且泛乳酸化水平随乳酸浓度梯度升高而增加。IGF2BP3敲低可降低组蛋白乳酸化修饰水平。通过整合RIP-seq和MeRIP-seq数据,鉴定出699个其转录本同时被IGF2BP3特异性结合且携带m6A修饰的候选基因,这些基因显著富集于表观遗传调控和代谢相关通路。EP300转录本上IGF2BP3的结合区域与m6A修饰位点高度重合,且EP300的表达水平在IGF2BP3敲低后明显下调。结论:外源性乳酸通过泛乳酸化修饰促进TNBC的恶性进展。IGF2BP3通过m6A依赖性机制调控EP300的表达,进而介导组蛋白乳酸化修饰与代谢重编程的协同作用,最终驱动TNBC的肿瘤进展。上述发现为靶向代谢-表观遗传交互调控的新型治疗策略提供了理论依据。展开更多
Background Resistance to traditional intense chemotherapy and high invasiveness are characteristics of head and neck squamous cell cancer(HNSCC).Numerous human disorders are linked to N6-methyladenosine(m6A)modificati...Background Resistance to traditional intense chemotherapy and high invasiveness are characteristics of head and neck squamous cell cancer(HNSCC).Numerous human disorders are linked to N6-methyladenosine(m6A)modification of RNA,and the genetic changes in m6A regulatory genes in HNSCC are not well-understood.There is also a pressing need to find efficient targets and inhibitors for the treatment of HNSCC.This investigation examined the RNA m6A alteration in HNSCC and found putative IGFBP3 inhibitors for potential use.Methods We examined m6A regulator gene expression data from the public Gene Expression Omnibus(GEO)database in both normal tissues and patient HNSCC.For bioinformatics analysis,the R package and additional tools,including the m6A2Target database,Gene Ontology(GO)functional and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses,Gene Expression Profiling Interactive Analysis(GEPIA),and Human Protein Atlas,were used to examine the molecular mechanisms and prognostic impact for regulators that are distinctly expressed.Furthermore,TCMBank molecules were employed for virtual screening to find possible inhibitors.Results Out of the 151 differentially expressed genes(DEGs)found in the chosen datasets,insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3)was the m6A regulator that was elevated in HNSCC.The GO and KEGG analyses revealed that the target genes of IGF2BP3 were mostly enriched in several pathways and activities linked to cancer.In patients with HNSCC,IGF2BP3 showed an effective predictive effect by GEPIA analysis.Virtual screening yielded four traditional Chinese medicine ingredients as putative IGF2BP3 inhibitors for additional research.Discussion This work learned the role and prognostic effect of IGF2BP3 in HNSCC;meanwhile,the potential inhibitors of IGF2BP3 were identified for further study.展开更多
BACKGROUND N6-methyladenosine(m6A)exerts a pro-carcinogenic effect in diverse cancers.The relationship between m6A-reading protein IGF2BP3 and gastric cancer(GC)has not yet been fully elucidated.AIM To investigate the...BACKGROUND N6-methyladenosine(m6A)exerts a pro-carcinogenic effect in diverse cancers.The relationship between m6A-reading protein IGF2BP3 and gastric cancer(GC)has not yet been fully elucidated.AIM To investigate the molecular mechanisms of IGF2BP3 in GC carcinogenesis and progression and thus provide a rationale for novel therapeutic strategies.METHODS Expression levels of IGF2BP3 in GC were determined using quantitative reverse transcription polymerase chain reaction(qRT-PCR),western blot(WB),and immunohistochemistry,and their associations with patients’clinicopathological characteristics were analyzed.The role of IGF2BP3 in GC was investigated using cellular functional assays and subcutaneous xenograft models,and its downstream targets and signaling pathways were identified using highthroughput sequencing,bioinformatics analysis,RNA immunoprecipitation qPCR,dual luciferase reporter assay,qRT-PCR,and WB.The mechanism of IGF2BP3 in GC was validated via WB and rescue and inhibition experiments.RESULTS IGF2BP3 was highly expressed in GC and associated with diffuse-type GC,incidence of lymph node metastasis,advanced tumor node metastasis stage,and deeper tumor invasion depth.In vitro experiments demonstrated that IGF2BP3 promoted proliferation,migration,and invasiveness of GC cells,while inhibiting apoptosis and augmenting intracellular levels of glucose metabolism.In vivo experiments revealed that IGF2BP3 contributes to the growth of GC.Mechanistically,IGF2BP3 recognized and bound to the m6A site at position 1427 on FBXO32 messenger RNA,thereby increasing protein expression of FBXO32,and further activated the downstream cyclic guanosine monophosphate-protein kinase G(cGMP-PKG)signaling pathway to modulate various biological functions of GC cells and promote progression of GC.Furthermore,treatment with a selective PKG inhibitor KT5823 significantly suppressed the proliferative capacity of GC cells.CONCLUSION IGF2BP3 increases FBXO32 protein expression in an m6A-dependent manner,activates the cGMP-PKG signaling pathway,and promotes GC progression.Targeting of the IGF2BP3/FBXO32/cGMP-PKG axis could thus represent a promising therapeutic modality for GC.展开更多
Numerous studies have characterized the critical role of circular RNAs(circRNAs)as regulatory factors in the progression of multiple cancers.However,the biological functions of circRNAs and their underlying molecular ...Numerous studies have characterized the critical role of circular RNAs(circRNAs)as regulatory factors in the progression of multiple cancers.However,the biological functions of circRNAs and their underlying molecular mechanisms in the progression of uveal melanoma(UM)remain enigmatic.In this study,we identified a novel circRNA,circ_0053943,through re-analysis of UM microarray data and quantitative RT-PCR.Circ_0053943 was found to be upregulated in UM and to promote the proliferation and metastatic ability of UM cells in both in vitro and in vivo settings.Mechanistically,circ_0053943 was observed to bind to the KH1 and KH2 domains of insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3),thereby enhancing the function of IGF2BP3 by stabilizing its target mRNA.RNA sequencing assays identified epidermal growth factor receptor(EGFR)as a target gene of circ_0053943 and IGF2BP3 at the transcriptional level.Rescue assays demonstrated that circ_0053943 exerts its biological function by stabilizing EGFR mRNA and regulating the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway.Collectively,circ_0053943 may promote UM progression by stabilizing EGFR mRNA and activating the MAPK/ERK signaling pathway through the formation of a circ_0053943/IGF2BP3/EGFR RNA-protein ternary complex,thus providing a potential biomarker and therapeutic target for UM.展开更多
文摘目的:探讨乳酸对三阴性乳腺癌(triple-negative breast cancer,TNBC)细胞恶性行为的影响,并揭示胰岛素样生长因子2 mRNA结合蛋白3(insulin-like growth factor 2 m RNA binding protein 3,IGF2BP3)通过m6A依赖性方式调控EP300,进而介导代谢重编程的表观遗传学机制。方法:通过外源性添加乳酸钠处理TNBC细胞,采用CCK-8、集落形成、划痕愈合和Transwell实验检测细胞增殖与迁移能力;利用shRNA慢病毒载体构建IGF2BP3稳定敲低细胞模型,结合Western blot分析组蛋白乳酸化修饰水平;进一步通过RIP-seq、MeRIP-seq筛选IGF2BP3的m6A靶基因,并利用qRT-PCR和Western blot验证关键分子表达。结果:乳酸钠处理显著增强了TNBC细胞的增殖和迁移能力,且泛乳酸化水平随乳酸浓度梯度升高而增加。IGF2BP3敲低可降低组蛋白乳酸化修饰水平。通过整合RIP-seq和MeRIP-seq数据,鉴定出699个其转录本同时被IGF2BP3特异性结合且携带m6A修饰的候选基因,这些基因显著富集于表观遗传调控和代谢相关通路。EP300转录本上IGF2BP3的结合区域与m6A修饰位点高度重合,且EP300的表达水平在IGF2BP3敲低后明显下调。结论:外源性乳酸通过泛乳酸化修饰促进TNBC的恶性进展。IGF2BP3通过m6A依赖性机制调控EP300的表达,进而介导组蛋白乳酸化修饰与代谢重编程的协同作用,最终驱动TNBC的肿瘤进展。上述发现为靶向代谢-表观遗传交互调控的新型治疗策略提供了理论依据。
基金National Natural Science Foundation of China(22307109),Natural Science Foundation of Henan Province(222300420510)Key Research Project Plan for Higher Education Institutions in Henan Province(23A350013)the Tackle Key Problems in Science and Technology Project of Henan Province,China(242102311226,242102310417).
文摘Background Resistance to traditional intense chemotherapy and high invasiveness are characteristics of head and neck squamous cell cancer(HNSCC).Numerous human disorders are linked to N6-methyladenosine(m6A)modification of RNA,and the genetic changes in m6A regulatory genes in HNSCC are not well-understood.There is also a pressing need to find efficient targets and inhibitors for the treatment of HNSCC.This investigation examined the RNA m6A alteration in HNSCC and found putative IGFBP3 inhibitors for potential use.Methods We examined m6A regulator gene expression data from the public Gene Expression Omnibus(GEO)database in both normal tissues and patient HNSCC.For bioinformatics analysis,the R package and additional tools,including the m6A2Target database,Gene Ontology(GO)functional and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses,Gene Expression Profiling Interactive Analysis(GEPIA),and Human Protein Atlas,were used to examine the molecular mechanisms and prognostic impact for regulators that are distinctly expressed.Furthermore,TCMBank molecules were employed for virtual screening to find possible inhibitors.Results Out of the 151 differentially expressed genes(DEGs)found in the chosen datasets,insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3)was the m6A regulator that was elevated in HNSCC.The GO and KEGG analyses revealed that the target genes of IGF2BP3 were mostly enriched in several pathways and activities linked to cancer.In patients with HNSCC,IGF2BP3 showed an effective predictive effect by GEPIA analysis.Virtual screening yielded four traditional Chinese medicine ingredients as putative IGF2BP3 inhibitors for additional research.Discussion This work learned the role and prognostic effect of IGF2BP3 in HNSCC;meanwhile,the potential inhibitors of IGF2BP3 were identified for further study.
基金Supported by the Hebei Natural Science Foundation,No.H2025206524,No.H2022206292 and No.H2024206140Hebei Provincial Government-funded Provincial Medical Excellent Talent Project,No.ZF2023025,No.ZF2024134 and No.LS202008+3 种基金Key RD Program of Hebei Province,No.223777103D and No.223777113DPrevention and Treatment of Geriatric Diseases by Hebei Provincial Department of Finance,No.LNB202202 and No.LNB201909Spark Scientific Research Project of the First Hospital of Hebei Medical University,No.XH202504,No.XH202312 and No.XH201805Hebei Province Medical Applicable Technology Tracking Project,No.G2019035.
文摘BACKGROUND N6-methyladenosine(m6A)exerts a pro-carcinogenic effect in diverse cancers.The relationship between m6A-reading protein IGF2BP3 and gastric cancer(GC)has not yet been fully elucidated.AIM To investigate the molecular mechanisms of IGF2BP3 in GC carcinogenesis and progression and thus provide a rationale for novel therapeutic strategies.METHODS Expression levels of IGF2BP3 in GC were determined using quantitative reverse transcription polymerase chain reaction(qRT-PCR),western blot(WB),and immunohistochemistry,and their associations with patients’clinicopathological characteristics were analyzed.The role of IGF2BP3 in GC was investigated using cellular functional assays and subcutaneous xenograft models,and its downstream targets and signaling pathways were identified using highthroughput sequencing,bioinformatics analysis,RNA immunoprecipitation qPCR,dual luciferase reporter assay,qRT-PCR,and WB.The mechanism of IGF2BP3 in GC was validated via WB and rescue and inhibition experiments.RESULTS IGF2BP3 was highly expressed in GC and associated with diffuse-type GC,incidence of lymph node metastasis,advanced tumor node metastasis stage,and deeper tumor invasion depth.In vitro experiments demonstrated that IGF2BP3 promoted proliferation,migration,and invasiveness of GC cells,while inhibiting apoptosis and augmenting intracellular levels of glucose metabolism.In vivo experiments revealed that IGF2BP3 contributes to the growth of GC.Mechanistically,IGF2BP3 recognized and bound to the m6A site at position 1427 on FBXO32 messenger RNA,thereby increasing protein expression of FBXO32,and further activated the downstream cyclic guanosine monophosphate-protein kinase G(cGMP-PKG)signaling pathway to modulate various biological functions of GC cells and promote progression of GC.Furthermore,treatment with a selective PKG inhibitor KT5823 significantly suppressed the proliferative capacity of GC cells.CONCLUSION IGF2BP3 increases FBXO32 protein expression in an m6A-dependent manner,activates the cGMP-PKG signaling pathway,and promotes GC progression.Targeting of the IGF2BP3/FBXO32/cGMP-PKG axis could thus represent a promising therapeutic modality for GC.
基金国家自然科学基金(82270162,82270224,82070178)北京市自然科学基金(7222175)+2 种基金军队卫勤保障能力创新与生成专项(21WQ034)保健专项科研课题重点项目(21BJZ30)国家重点研发计划(2021Y FA 1100904)。
文摘目的:探寻IGF2BP3基因表达水平与急性髓系白血病(AML)患者预后的关系。方法:通过对本中心27例AML患者骨髓原代白血病细胞进行转录组高通量测序,分析IGF2BP3基因表达水平与患者临床特征之间的关系,并在初治AML患者及难治AML(Refractory AML)患者样本中验证。分析20例健康对照者和26例AML患者中IGF2BP3基因表达水平的差异。采用RT-qPCR、Western blot检测两种蒽环类耐药细胞系(HL60/ADR、K562/ADR)中IGF2BP3表达水平,比较其与敏感细胞(HL60、K562)的表达差异。通过3个数据集,分析IGF2BP3在AML患者中的表达水平及与预后的关系,进一步使用Cox生存分析IGF2BP3在AML中的预后价值。结果:在本中心27例AML患者骨髓原代白血病细胞中,难治性AML患者的IGF2BP3表达量明显高于化疗敏感的患者(P=0.0343),白血病细胞髓外浸润(extramedullary infiltration,EMI)患者的IGF2BP3表达量明显高于无髓外浸润的AML患者(P=0.0049)。与健康人比较,IGF2BP3在AML患者中表达增加(P=0.0009)。蒽环类耐药细胞系(HL60/ADR、K562/ADR)中IGF2BP3 mRNA的表达显著高于敏感细胞系(K562/ADR vs K562,P=0.0430;HL60/ADR vs HL60,P=0.7369)。Western blot结果显示,耐药细胞中IGF2BP3蛋白表达显著高于敏感细胞(P<0.001)。qPCR结果显示,难治AML患者中IGF2BP3的mRNA表达水平明显高于化疗敏感患者(P=0.002)。在3个大样本AML患者队列中IGF2BP3高表达预示AML预后不良(P<0.05)。单因素和多因素预后分析证实IGF2BP3高表达与患者较短的无事件生存(HR=1.887,P=0.024)和总体生存(HR=1.619,P=0.016)显著相关。结论:IGF2BP3基因高表达可能是AML预后不良的重要因素,提示IGF2BP3基因有望成为AML的临床预后评估和提供治疗策略的新的分子标志物。
基金supported by the National Natural Science Foundation of China(Nos.82273159 and 82171838)the Jiangsu Province’s Science and Technology Project(No.BE2020722).
文摘Numerous studies have characterized the critical role of circular RNAs(circRNAs)as regulatory factors in the progression of multiple cancers.However,the biological functions of circRNAs and their underlying molecular mechanisms in the progression of uveal melanoma(UM)remain enigmatic.In this study,we identified a novel circRNA,circ_0053943,through re-analysis of UM microarray data and quantitative RT-PCR.Circ_0053943 was found to be upregulated in UM and to promote the proliferation and metastatic ability of UM cells in both in vitro and in vivo settings.Mechanistically,circ_0053943 was observed to bind to the KH1 and KH2 domains of insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3),thereby enhancing the function of IGF2BP3 by stabilizing its target mRNA.RNA sequencing assays identified epidermal growth factor receptor(EGFR)as a target gene of circ_0053943 and IGF2BP3 at the transcriptional level.Rescue assays demonstrated that circ_0053943 exerts its biological function by stabilizing EGFR mRNA and regulating the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway.Collectively,circ_0053943 may promote UM progression by stabilizing EGFR mRNA and activating the MAPK/ERK signaling pathway through the formation of a circ_0053943/IGF2BP3/EGFR RNA-protein ternary complex,thus providing a potential biomarker and therapeutic target for UM.
