目的分析三阴性乳腺癌(triple negative breast cancer,TNBC)细胞敲低YTHDF3(YT521-B homology domain family 3)前后的蛋白质表达差异,探究YTHDF3调控TNBC细胞蛋白质功能的可能机制。方法利用分子克隆、靶点设计和病毒载体包装技术构建...目的分析三阴性乳腺癌(triple negative breast cancer,TNBC)细胞敲低YTHDF3(YT521-B homology domain family 3)前后的蛋白质表达差异,探究YTHDF3调控TNBC细胞蛋白质功能的可能机制。方法利用分子克隆、靶点设计和病毒载体包装技术构建YTHDF3稳定敲低的MDA-MB-231细胞系并采用Western blot法进一步验证。裂解细胞蛋白,采用超高效液相色谱(ultra performance liquid chromatography,UPLC)和液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)方法获取因敲低YTHDF3而出现的差异表达蛋白。使用String数据库及Cytoscape 3.10.1软件,绘制并分析蛋白质互作网络(protein-protein interaction,PPI)并筛选核心靶点。通过David数据库对核心靶点进行京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路和基因本体(gene ontology,GO)功能富集分析。采用RNA干扰技术分别敲低MDA-MB-231和HS-578T细胞中的YTHDF3或UUbiquitin(BBB)基因,Western blot法验证YTHDF3敲低的两种TNBC细胞中核心靶蛋白的表达,并采用Transwell法检测UBB敲低对TNBC细胞侵袭和迁移的影响。结果成功构建了YTHDF3稳定敲低的TNBC细胞模型,并在该细胞中定量到2295个蛋白,筛选出261个差异表达蛋白(126种下调和135种上调)。GO分析结果显示,差异下调蛋白主要涉及翻译和蛋白质稳定性,而对细胞群体增殖的负调控蛋白则是上调表达。KEGG结果表明,差异下调蛋白主要富集于剪接体相关信号通路,而差异上调蛋白主要与凋亡通路相关。PPI分析表明有6个核心靶点(RPS27A、UBA52、UBC、UBB、RPL4和RPS2)下调。Western blot分析揭示YTHDF3敲低的MDA-MB-231和HS-578T细胞中YTHDF3和UBB蛋白表达均降低。Transwell结果指出UBB缺失抑制TNBC细胞侵袭和迁移水平。结论本文以全新视角的研究,揭示了YTHDF3通过调控下游核心靶点UBB在TNBC细胞侵袭转移中发挥着重要作用。展开更多
Liver injury is a major health issue with significant implications for liver function and overall well-being,but precise mechanisms of the N^(6-)methyladenine(m^(6)A)reader YTHDF3 in liver injury remain severely under...Liver injury is a major health issue with significant implications for liver function and overall well-being,but precise mechanisms of the N^(6-)methyladenine(m^(6)A)reader YTHDF3 in liver injury remain severely understudied.Here,we discovered that Ythdf3 knockout exacerbated CCL4-induced liver injury with a reduction in functional hepatocytes and liver stem cells using single cell RNA-sequencing and organoid culture.Furthermore,Mettl14 and YTHDF3-dependent RNA m^(6)A dysregulation induced DNA damage.Moreover,we found YTHDF3 could bind and modulate CCAAT/enhancer-binding protein-alpha(CEBPA)translation in an m^(6)A-dependent manner.Mechanistically,knockout of Ythdf3 impeded the translation of CEBPA,subsequently inhibiting the expression of poly(ADP-ribose)(PAR)polymerase-1(PARP1)and Peroxiredoxin 2(PRDX2).This inhibition promoted DNA damage and genomic instability,ultimately exacerbating liver damage.This work uncovers an essential role of m^(6)A/YTHDF3/CEBPA regulatory axes in governing cell fates and genomic stability,thereby preventing liver injury.Importantly,these findings offer potential therapeutic avenues for targeting YTHDF3 and CEBPA in the treatment of liver injuryrelated diseases.展开更多
文摘目的分析三阴性乳腺癌(triple negative breast cancer,TNBC)细胞敲低YTHDF3(YT521-B homology domain family 3)前后的蛋白质表达差异,探究YTHDF3调控TNBC细胞蛋白质功能的可能机制。方法利用分子克隆、靶点设计和病毒载体包装技术构建YTHDF3稳定敲低的MDA-MB-231细胞系并采用Western blot法进一步验证。裂解细胞蛋白,采用超高效液相色谱(ultra performance liquid chromatography,UPLC)和液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)方法获取因敲低YTHDF3而出现的差异表达蛋白。使用String数据库及Cytoscape 3.10.1软件,绘制并分析蛋白质互作网络(protein-protein interaction,PPI)并筛选核心靶点。通过David数据库对核心靶点进行京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路和基因本体(gene ontology,GO)功能富集分析。采用RNA干扰技术分别敲低MDA-MB-231和HS-578T细胞中的YTHDF3或UUbiquitin(BBB)基因,Western blot法验证YTHDF3敲低的两种TNBC细胞中核心靶蛋白的表达,并采用Transwell法检测UBB敲低对TNBC细胞侵袭和迁移的影响。结果成功构建了YTHDF3稳定敲低的TNBC细胞模型,并在该细胞中定量到2295个蛋白,筛选出261个差异表达蛋白(126种下调和135种上调)。GO分析结果显示,差异下调蛋白主要涉及翻译和蛋白质稳定性,而对细胞群体增殖的负调控蛋白则是上调表达。KEGG结果表明,差异下调蛋白主要富集于剪接体相关信号通路,而差异上调蛋白主要与凋亡通路相关。PPI分析表明有6个核心靶点(RPS27A、UBA52、UBC、UBB、RPL4和RPS2)下调。Western blot分析揭示YTHDF3敲低的MDA-MB-231和HS-578T细胞中YTHDF3和UBB蛋白表达均降低。Transwell结果指出UBB缺失抑制TNBC细胞侵袭和迁移水平。结论本文以全新视角的研究,揭示了YTHDF3通过调控下游核心靶点UBB在TNBC细胞侵袭转移中发挥着重要作用。
基金supported by the National Key Research and Development Program of China(2022YFD130040307)the Fundamental Research Funds for the Central Universities(KYCXJC2024001)the National Institute of Mental Health(R01 MH122142,B.C.D.)of the National Institutes of Health(NIH)。
文摘Liver injury is a major health issue with significant implications for liver function and overall well-being,but precise mechanisms of the N^(6-)methyladenine(m^(6)A)reader YTHDF3 in liver injury remain severely understudied.Here,we discovered that Ythdf3 knockout exacerbated CCL4-induced liver injury with a reduction in functional hepatocytes and liver stem cells using single cell RNA-sequencing and organoid culture.Furthermore,Mettl14 and YTHDF3-dependent RNA m^(6)A dysregulation induced DNA damage.Moreover,we found YTHDF3 could bind and modulate CCAAT/enhancer-binding protein-alpha(CEBPA)translation in an m^(6)A-dependent manner.Mechanistically,knockout of Ythdf3 impeded the translation of CEBPA,subsequently inhibiting the expression of poly(ADP-ribose)(PAR)polymerase-1(PARP1)and Peroxiredoxin 2(PRDX2).This inhibition promoted DNA damage and genomic instability,ultimately exacerbating liver damage.This work uncovers an essential role of m^(6)A/YTHDF3/CEBPA regulatory axes in governing cell fates and genomic stability,thereby preventing liver injury.Importantly,these findings offer potential therapeutic avenues for targeting YTHDF3 and CEBPA in the treatment of liver injuryrelated diseases.
