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
F-actin microstructures dominate cellular viscoelasticity and have been used to identify the migration and malignance of living cancer cells.Diabetic cancer patients suffer from increased metastasis and tumor recurren...F-actin microstructures dominate cellular viscoelasticity and have been used to identify the migration and malignance of living cancer cells.Diabetic cancer patients suffer from increased metastasis and tumor recurrence.However,the long-term evolution and correlation of F-actin microstructures and viscoelasticity distribution are still poorly understood in living cancer cells under varying glucose environment.Herein,by using atomic force microscopy with amplitude modulation-frequency modulation and nanoindentation mode,we characterized the hierarchical F-actin microstructures and the multi-passage viscoelasticity evolution in living Huh-7 cancer cells transferred from high to low glucose level.The highly oriented stress fibers connected by thinner fiber networks were observed in high glucose environment.The circumferential actin networks composed by straight segment-like fibers and the randomly distributed actin fragments connected by ultrathin crosslinking fibers were observed in low glucose environment.The viscoelasticity within the nucleus and the cytoplasm of living Huh-7 cancer cells showed longterm fluctuations over tens of passages after switching glucose environments.The viscoelasticity of cytoplasm was more responsive to the change of glucose environments than nucleus,which was due to the reorganization of F-actin microstructures.Our work provides the microstructural and nanomechanical understanding on the migration and proliferation of living cancer cells under varying glucose environment.展开更多
目的研究Plectin的表达与肝癌细胞迁移能力的关系,揭示Plectin表达影响肝癌细胞迁移行为的分子机理。方法首先,Western blot检测正常肝细胞和肝癌细胞中Plectin的表达。其次,构建Plectin下调的肝癌细胞株,设立对照组(shNC组)和shPLEC组...目的研究Plectin的表达与肝癌细胞迁移能力的关系,揭示Plectin表达影响肝癌细胞迁移行为的分子机理。方法首先,Western blot检测正常肝细胞和肝癌细胞中Plectin的表达。其次,构建Plectin下调的肝癌细胞株,设立对照组(shNC组)和shPLEC组,各组分别设溶剂对照组(shNC+DMSO组或shPLEC+DMSO组)和F-actin骨架聚合诱导剂Jasplakinolide组(shNC+Jasp组或shPLEC+Jasp组)。采用Western blot检测各组肝癌细胞中Plectin的表达及上皮-间质转化(epithelial-mesenchymal transition,EMT)相关分子(N-cadherin、vimentin和E-cadherin)的表达;采用Transwell小室法分析肝癌细胞的迁移能力;采用KEGG(Kyoto Encyclopedia of Genes and Genomes)分析与Plectin基因有关的信号通路;采用免疫荧光技术检测Plectin表达变化对细胞骨架F-actin聚合的影响。结果与正常肝细胞相比,Plectin在肝癌细胞中高表达。与shNC组相比,shPLEC组Plectin的表达降低(P<0.05),肝癌细胞的迁移能力减弱(P<0.05),EMT进程被抑制(N-cadherin和vimentin表达降低,E-cadherin表达升高)(P<0.05);KEGG分析发现细胞骨架F-actin调控与Plectin的联系最为密切,shPLEC组肝癌细胞骨架F-actin发生解聚。采用F-actin骨架聚合诱导剂Jasplakinolide处理后,与shPLEC+DMSO组相比,shPLEC+Jasp组肝癌细胞迁移能力增强(P<0.05),EMT进程有所恢复(N-cadherin和vimentin表达升高,E-cadherin表达降低)(P<0.05),同时肝癌细胞骨架F-actin聚合亦有所恢复。结论Plectin在肝癌细胞中高表达,肝癌细胞中Plectin通过诱导F-actin聚合促进肝癌细胞的迁移和EMT。展开更多
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.
基金supported by the National Natural Science Foundation of China(Grant No.11972383)to Wenpeng Zhuby the National Natural Science Foundation of China(Grant No.12132020)to Yue Zhengby the Guangdong Provincial Key Laboratory of Magnetoelectric Physics and Devices(Grant No.2022B1212010008).
文摘F-actin microstructures dominate cellular viscoelasticity and have been used to identify the migration and malignance of living cancer cells.Diabetic cancer patients suffer from increased metastasis and tumor recurrence.However,the long-term evolution and correlation of F-actin microstructures and viscoelasticity distribution are still poorly understood in living cancer cells under varying glucose environment.Herein,by using atomic force microscopy with amplitude modulation-frequency modulation and nanoindentation mode,we characterized the hierarchical F-actin microstructures and the multi-passage viscoelasticity evolution in living Huh-7 cancer cells transferred from high to low glucose level.The highly oriented stress fibers connected by thinner fiber networks were observed in high glucose environment.The circumferential actin networks composed by straight segment-like fibers and the randomly distributed actin fragments connected by ultrathin crosslinking fibers were observed in low glucose environment.The viscoelasticity within the nucleus and the cytoplasm of living Huh-7 cancer cells showed longterm fluctuations over tens of passages after switching glucose environments.The viscoelasticity of cytoplasm was more responsive to the change of glucose environments than nucleus,which was due to the reorganization of F-actin microstructures.Our work provides the microstructural and nanomechanical understanding on the migration and proliferation of living cancer cells under varying glucose environment.
文摘目的研究Plectin的表达与肝癌细胞迁移能力的关系,揭示Plectin表达影响肝癌细胞迁移行为的分子机理。方法首先,Western blot检测正常肝细胞和肝癌细胞中Plectin的表达。其次,构建Plectin下调的肝癌细胞株,设立对照组(shNC组)和shPLEC组,各组分别设溶剂对照组(shNC+DMSO组或shPLEC+DMSO组)和F-actin骨架聚合诱导剂Jasplakinolide组(shNC+Jasp组或shPLEC+Jasp组)。采用Western blot检测各组肝癌细胞中Plectin的表达及上皮-间质转化(epithelial-mesenchymal transition,EMT)相关分子(N-cadherin、vimentin和E-cadherin)的表达;采用Transwell小室法分析肝癌细胞的迁移能力;采用KEGG(Kyoto Encyclopedia of Genes and Genomes)分析与Plectin基因有关的信号通路;采用免疫荧光技术检测Plectin表达变化对细胞骨架F-actin聚合的影响。结果与正常肝细胞相比,Plectin在肝癌细胞中高表达。与shNC组相比,shPLEC组Plectin的表达降低(P<0.05),肝癌细胞的迁移能力减弱(P<0.05),EMT进程被抑制(N-cadherin和vimentin表达降低,E-cadherin表达升高)(P<0.05);KEGG分析发现细胞骨架F-actin调控与Plectin的联系最为密切,shPLEC组肝癌细胞骨架F-actin发生解聚。采用F-actin骨架聚合诱导剂Jasplakinolide处理后,与shPLEC+DMSO组相比,shPLEC+Jasp组肝癌细胞迁移能力增强(P<0.05),EMT进程有所恢复(N-cadherin和vimentin表达升高,E-cadherin表达降低)(P<0.05),同时肝癌细胞骨架F-actin聚合亦有所恢复。结论Plectin在肝癌细胞中高表达,肝癌细胞中Plectin通过诱导F-actin聚合促进肝癌细胞的迁移和EMT。
