目的分析甲基转移酶3(METTL3)抑制剂STM2457对人肝癌细胞系HepG2的影响,重点研究其对N6-甲基腺苷(m6A)表达的影响及其抗肿瘤机制。方法将HepG2细胞分为实验组(STM2457处理)和对照组(DMSO处理)。利用纳米孔(Nanopore)测序技术,结合m6Anet...目的分析甲基转移酶3(METTL3)抑制剂STM2457对人肝癌细胞系HepG2的影响,重点研究其对N6-甲基腺苷(m6A)表达的影响及其抗肿瘤机制。方法将HepG2细胞分为实验组(STM2457处理)和对照组(DMSO处理)。利用纳米孔(Nanopore)测序技术,结合m6Anet,NanoCount,xPore和GFOLD方法,分别对m6A修饰水平、转录组表达及差异基因进行分析。通过基因本体(GO)和京都基因与基因组百科(KEGG)对差异基因进行功能富集分析。结果STM2457降低HepG2细胞的m6A修饰位点数量(6446 vs 11549)及修饰水平(0.95±0.03 vs 0.98±0.03),差异具有统计学意义(Z=-19.915,P<0.01)。差异基因分析共筛选出109个上调基因和340个下调基因,其中与肝癌发生发展密切相关的基因PDLIM5、AZGP1和RNASET2,其m6A修饰水平降低,而基因表达水平升高。功能富集分析结果显示,差异基因主要富集在细胞黏附、凋亡、翻译调控及肝细胞癌相关通路。结论STM2457通过抑制METTL3活性,降低HepG2细胞的m6A修饰水平,上调基因PDLIM5,AZGP1和RNASET2的表达,促进HepG2细胞凋亡,为肝癌治疗提供潜在治疗靶点。展开更多
DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a hug...DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample.In this work,we proposed a protospacer adjacent motif(PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method.Through recognizing the ds DNA with toehold region,CRISPR-Cas12a not only got rid of the limitation of PAM,but also improved its distinction ability for single Cp G site methylation,nearly 5-fold that of conventional PAM-containing ds DNA.We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single Cp G methylation site.Our results showed that the discrimination factor was >200.Then,we constructed toe-ds DNA by using “heating and freezing”,which made our method universally applicable and feasible.In addition,we greatly simplified the difficulty of primer design.Our method detected four highly methylated genes acyl carrier protein(ACP),CLV3/ESR-related(CLE),Disabled(DAB) and Homeobox(HOX) with a detection limit of 0.01 % and excellent linearity in DNA methylation standards.Then,we verified the clinical utility of this method in 29 hepatocellular carcinomas,11 ovarian cancers and4 health people.In conclusion,we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method,which achieves high congruence in sensitivity,specificity and universality,fully demonstrating its significant clinical application value.展开更多
文摘目的分析甲基转移酶3(METTL3)抑制剂STM2457对人肝癌细胞系HepG2的影响,重点研究其对N6-甲基腺苷(m6A)表达的影响及其抗肿瘤机制。方法将HepG2细胞分为实验组(STM2457处理)和对照组(DMSO处理)。利用纳米孔(Nanopore)测序技术,结合m6Anet,NanoCount,xPore和GFOLD方法,分别对m6A修饰水平、转录组表达及差异基因进行分析。通过基因本体(GO)和京都基因与基因组百科(KEGG)对差异基因进行功能富集分析。结果STM2457降低HepG2细胞的m6A修饰位点数量(6446 vs 11549)及修饰水平(0.95±0.03 vs 0.98±0.03),差异具有统计学意义(Z=-19.915,P<0.01)。差异基因分析共筛选出109个上调基因和340个下调基因,其中与肝癌发生发展密切相关的基因PDLIM5、AZGP1和RNASET2,其m6A修饰水平降低,而基因表达水平升高。功能富集分析结果显示,差异基因主要富集在细胞黏附、凋亡、翻译调控及肝细胞癌相关通路。结论STM2457通过抑制METTL3活性,降低HepG2细胞的m6A修饰水平,上调基因PDLIM5,AZGP1和RNASET2的表达,促进HepG2细胞凋亡,为肝癌治疗提供潜在治疗靶点。
基金financially supported by the Natural Science Foundation of Wuhan City (Chenguang Project) (No.2024040801020331)the Natural Science Foundation of Hubei Province of China (No.2023AFB402)+1 种基金the National Key Research and Development Program of China (No.2023YFE0210200)Interdisciplinary Research Program of HUST。
文摘DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample.In this work,we proposed a protospacer adjacent motif(PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method.Through recognizing the ds DNA with toehold region,CRISPR-Cas12a not only got rid of the limitation of PAM,but also improved its distinction ability for single Cp G site methylation,nearly 5-fold that of conventional PAM-containing ds DNA.We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single Cp G methylation site.Our results showed that the discrimination factor was >200.Then,we constructed toe-ds DNA by using “heating and freezing”,which made our method universally applicable and feasible.In addition,we greatly simplified the difficulty of primer design.Our method detected four highly methylated genes acyl carrier protein(ACP),CLV3/ESR-related(CLE),Disabled(DAB) and Homeobox(HOX) with a detection limit of 0.01 % and excellent linearity in DNA methylation standards.Then,we verified the clinical utility of this method in 29 hepatocellular carcinomas,11 ovarian cancers and4 health people.In conclusion,we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method,which achieves high congruence in sensitivity,specificity and universality,fully demonstrating its significant clinical application value.
