为分析E3泛素连接酶环指蛋白125(ring finger protein 125,RNF125)在肝癌中的表达及临床预后意义,并探究RNF125对肝癌侵袭和转移的影响及其作用机制,通过ENCORI数据库和临床组织样本分析RNF125在肝癌和癌旁组织中的表达量,使用ENCORI数...为分析E3泛素连接酶环指蛋白125(ring finger protein 125,RNF125)在肝癌中的表达及临床预后意义,并探究RNF125对肝癌侵袭和转移的影响及其作用机制,通过ENCORI数据库和临床组织样本分析RNF125在肝癌和癌旁组织中的表达量,使用ENCORI数据库分析RNF125与肝癌预后的关系。构建敲低和过表达RNF125的肝癌细胞系,通过Transwell试验和细胞划痕试验检验RNF125表达水平对肝癌细胞迁移、侵袭能力的影响。通过动物试验验证调控RNF125对体内肿瘤生长情况及对肿瘤免疫微环境的影响,质谱分析寻找RNF125的靶蛋白,qPCR、蛋白质免疫印迹技术、泛素化试验验证RNF125与MYH9之间的关系,通过蛋白质免疫印迹和细胞免疫荧光技术确定调控RNF125与MYH9对EMT标志蛋白的影响。结果表明:RNF125在肝癌组织中表达较低,RNF125的高表达提示良好的预后。RNF125的表达水平与肝癌细胞迁移和侵袭能力呈负相关。过表达RNF125使肿瘤生长延迟并增加免疫细胞的浸润水平。RNF125特异性结合MYH9并促进其泛素化降解。RNF125与上皮细胞标志物的表达水平呈正相关,与间充质细胞标志物呈负相关。过表达MYH9可逆转RNF125对EMT标志蛋白的影响。综上,RNF125在肝癌中表达下调,RNF125的高表达与肝癌患者的良好预后相关,RNF125通过泛素化MYH9来抑制肝癌的EMT过程,从而抑制肝癌的迁移和侵袭。展开更多
AIM:To explore the role of a previously-found MYH9 tail domain mutation(p.E1384Q)in the pathogenesis of congenital cataract.METHODS:The cell experiments were conducted in vitro.Wild-type(WT)MYH9 and p.E1384Q mutant fr...AIM:To explore the role of a previously-found MYH9 tail domain mutation(p.E1384Q)in the pathogenesis of congenital cataract.METHODS:The cell experiments were conducted in vitro.Wild-type(WT)MYH9 and p.E1384Q mutant fragments were constructed,which was then transiently transfected into Hek293T cell lines.Western blotting and quantitative real time polymerase chain reaction(qRT-PCR)were used to analyze the protein and mRNA level of non-muscle myosin IIA(NM IIA)and F-actin in transfected cells,and fluorescence microscopy was applied to explore the subcellular localization of NM IIA and F-actin.Cell counting kit-8(CCK8),woundhealing and double staining flow cytometry assays were performed to evaluate the proliferation,migration and apoptosis function of transfected cells,respectively.Transmission electron microscope was conducted to observe the alteration of organelle structure.RESULTS:The transiently-transfected WT and p.E1384Q mutant Hek293T cell lines was constructed.Western blot demonstrated that,comparing with MYH9WT group,the relative protein amount of NM IIA and F-actin significantly decreased in MYH9E1384Q cells(P<0.001).qRT-PCR analysis revealed that the relative mRNA amount of NM IIA and F-actin also significantly reduced in MYH9E1384Q cells when compared with MYH9WT.The immunofluorescence microscopy showed that the fluorescence signal of NM IIA and F-actin significantly decreased in E1384Q cells.The diffuse cytoplasmic distribution of NM IIA in MYH9WT was changed to be clumped distribution,presenting a“speckled”pattern characterized by aggregates of small size in MYH9E1384Q.Functional study revealed that the E1384Q mutation significantly inhibited cell proliferation(P=0.003)and migration(P<0.001),and promoted apoptosis(P<0.001).Electron microscope showed that the mutation remarkably decreased the number of mitochondria(P<0.001)and changed the phenotype of mitochondria.CONCLUSION:The missense gene mutation in MYH9(p.E1384Q)causing congenital cataract results in decreased amount and altered subcellular distribution of NM IIA and F-actin,accompanied by decreased cell proliferation and migration,promotes apoptosis and mitochondrial alteration.展开更多
文摘为分析E3泛素连接酶环指蛋白125(ring finger protein 125,RNF125)在肝癌中的表达及临床预后意义,并探究RNF125对肝癌侵袭和转移的影响及其作用机制,通过ENCORI数据库和临床组织样本分析RNF125在肝癌和癌旁组织中的表达量,使用ENCORI数据库分析RNF125与肝癌预后的关系。构建敲低和过表达RNF125的肝癌细胞系,通过Transwell试验和细胞划痕试验检验RNF125表达水平对肝癌细胞迁移、侵袭能力的影响。通过动物试验验证调控RNF125对体内肿瘤生长情况及对肿瘤免疫微环境的影响,质谱分析寻找RNF125的靶蛋白,qPCR、蛋白质免疫印迹技术、泛素化试验验证RNF125与MYH9之间的关系,通过蛋白质免疫印迹和细胞免疫荧光技术确定调控RNF125与MYH9对EMT标志蛋白的影响。结果表明:RNF125在肝癌组织中表达较低,RNF125的高表达提示良好的预后。RNF125的表达水平与肝癌细胞迁移和侵袭能力呈负相关。过表达RNF125使肿瘤生长延迟并增加免疫细胞的浸润水平。RNF125特异性结合MYH9并促进其泛素化降解。RNF125与上皮细胞标志物的表达水平呈正相关,与间充质细胞标志物呈负相关。过表达MYH9可逆转RNF125对EMT标志蛋白的影响。综上,RNF125在肝癌中表达下调,RNF125的高表达与肝癌患者的良好预后相关,RNF125通过泛素化MYH9来抑制肝癌的EMT过程,从而抑制肝癌的迁移和侵袭。
基金Supported by Beijing Municipal Natural Science Foundation(No.7202229No.7242168)China Primary Health Care Foundation(No.MTP2022C025).
文摘AIM:To explore the role of a previously-found MYH9 tail domain mutation(p.E1384Q)in the pathogenesis of congenital cataract.METHODS:The cell experiments were conducted in vitro.Wild-type(WT)MYH9 and p.E1384Q mutant fragments were constructed,which was then transiently transfected into Hek293T cell lines.Western blotting and quantitative real time polymerase chain reaction(qRT-PCR)were used to analyze the protein and mRNA level of non-muscle myosin IIA(NM IIA)and F-actin in transfected cells,and fluorescence microscopy was applied to explore the subcellular localization of NM IIA and F-actin.Cell counting kit-8(CCK8),woundhealing and double staining flow cytometry assays were performed to evaluate the proliferation,migration and apoptosis function of transfected cells,respectively.Transmission electron microscope was conducted to observe the alteration of organelle structure.RESULTS:The transiently-transfected WT and p.E1384Q mutant Hek293T cell lines was constructed.Western blot demonstrated that,comparing with MYH9WT group,the relative protein amount of NM IIA and F-actin significantly decreased in MYH9E1384Q cells(P<0.001).qRT-PCR analysis revealed that the relative mRNA amount of NM IIA and F-actin also significantly reduced in MYH9E1384Q cells when compared with MYH9WT.The immunofluorescence microscopy showed that the fluorescence signal of NM IIA and F-actin significantly decreased in E1384Q cells.The diffuse cytoplasmic distribution of NM IIA in MYH9WT was changed to be clumped distribution,presenting a“speckled”pattern characterized by aggregates of small size in MYH9E1384Q.Functional study revealed that the E1384Q mutation significantly inhibited cell proliferation(P=0.003)and migration(P<0.001),and promoted apoptosis(P<0.001).Electron microscope showed that the mutation remarkably decreased the number of mitochondria(P<0.001)and changed the phenotype of mitochondria.CONCLUSION:The missense gene mutation in MYH9(p.E1384Q)causing congenital cataract results in decreased amount and altered subcellular distribution of NM IIA and F-actin,accompanied by decreased cell proliferation and migration,promotes apoptosis and mitochondrial alteration.
