目的:构建高效的慢病毒GV115-AIF si RNA重组表达系统。方法:根据目的基因AIF以及RNA干扰序列设计原则,利用设计软件设计了3个可能的AIF si RNA序列。应用全基因合成技术和亚克隆技术构建GV115-AIF si RNA重组质粒,并采用聚合酶链反应技...目的:构建高效的慢病毒GV115-AIF si RNA重组表达系统。方法:根据目的基因AIF以及RNA干扰序列设计原则,利用设计软件设计了3个可能的AIF si RNA序列。应用全基因合成技术和亚克隆技术构建GV115-AIF si RNA重组质粒,并采用聚合酶链反应技术(PCR技术)和基因测序技术对GV115-AIF si RNA重组质粒鉴定。利用GV115病毒包装辅助p Helper 1.0质粒和p Helper 2.0质粒进行病毒包装。病毒包装后转染人胚肾293T细胞,通过应用逆转录定量PCR技术(RT-PCR技术)检测转染后对人胚肾293T细胞中AIF基因的敲减效率,筛选最高效的AIF si RNA基因序列。结果:GV115-AIF si RNA质粒PCR鉴定显示位于341bp附近的条带。测序结果与设计的基因序列完全吻合。3个可能的AIF si RNA序列中基因敲减效率最高的可达到88.3%。结论:成功构建高效的慢病毒GV115-AIF si RNA重组表达系统。展开更多
目的·挖掘F-box蛋白22(F-box only protein 22,FBXO22)的底物并明确其作用机制,进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子(apoptosis-inducing fac...目的·挖掘F-box蛋白22(F-box only protein 22,FBXO22)的底物并明确其作用机制,进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子(apoptosis-inducing factor,AIF)。采用变性条件下泛素化修饰实验检测FBXO22对AIF泛素化修饰的影响。通过短发夹RNA敲低FBXO22、过表达FBXO22检测AIF的表达水平。利用流式细胞仪检测FBXO22敲低或过表达对结肠癌细胞凋亡的影响。结果·免疫沉淀联合质谱分析显示FBXO22可与AIF结合,变性条件下泛素化修饰实验显示FBXO22可介导AIF的泛素化修饰。敲低FBXO22可上调AIF水平,过表达FBXO22可下调AIF水平。流式细胞术结果显示,敲低FBXO22可促进由甲基硝基亚硝基胍(N-methyl-N’-nitro-N-nitrosoguanidine,MNNG)等诱导的结肠癌细胞凋亡,过表达FBXO22则可抑制MNNG等诱导的结肠癌细胞凋亡。结论·FBXO22通过泛素化修饰降解AIF进而调控由AIF介导的结肠癌细胞凋亡。展开更多
Auditory neuropathy spectrum disorder(ANSD)represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function,but with the preservation of outer hair ce...Auditory neuropathy spectrum disorder(ANSD)represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function,but with the preservation of outer hair cell function.ANSD represents up to 15%of individuals with hearing impairments.Through mutation screening,bioinformatic analysis and expression studies,we have previously identified several apoptosis-inducing factor(AIF)mitochondria-associated 1(AIFM1)variants in ANSD families and in some other sporadic cases.Here,to elucidate the pathogenic mechanisms underlying each AIFM1 variant,we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system and constructed AIF-wild type(WT)and AIF-mutant(mut)(p.T260A,p.R422W,and p.R451Q)stable transfection cell lines.We then analyzed AIF structure,coenzyme-binding affinity,apoptosis,and other aspects.Results revealed that these variants resulted in impaired dimerization,compromising AIF function.The reduction reaction of AIF variants had proceeded slower than that of AIF-WT.The average levels of AIF dimerization in AIF variant cells were only 34.5%-49.7%of that of AIF-WT cells,resulting in caspase-independent apoptosis.The average percentage of apoptotic cells in the variants was 12.3%-17.9%,which was significantly higher than that(6.9%-7.4%)in controls.However,nicotinamide adenine dinucleotide(NADH)treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells.Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD,and introduce NADH as a potential drug for ANSD treatment.Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.展开更多
文摘目的:构建高效的慢病毒GV115-AIF si RNA重组表达系统。方法:根据目的基因AIF以及RNA干扰序列设计原则,利用设计软件设计了3个可能的AIF si RNA序列。应用全基因合成技术和亚克隆技术构建GV115-AIF si RNA重组质粒,并采用聚合酶链反应技术(PCR技术)和基因测序技术对GV115-AIF si RNA重组质粒鉴定。利用GV115病毒包装辅助p Helper 1.0质粒和p Helper 2.0质粒进行病毒包装。病毒包装后转染人胚肾293T细胞,通过应用逆转录定量PCR技术(RT-PCR技术)检测转染后对人胚肾293T细胞中AIF基因的敲减效率,筛选最高效的AIF si RNA基因序列。结果:GV115-AIF si RNA质粒PCR鉴定显示位于341bp附近的条带。测序结果与设计的基因序列完全吻合。3个可能的AIF si RNA序列中基因敲减效率最高的可达到88.3%。结论:成功构建高效的慢病毒GV115-AIF si RNA重组表达系统。
文摘目的·挖掘F-box蛋白22(F-box only protein 22,FBXO22)的底物并明确其作用机制,进一步探讨FBXO22在肿瘤细胞凋亡中的作用。方法·利用免疫沉淀联合质谱分析寻找并验证FBXO22的相互作用蛋白凋亡诱导因子(apoptosis-inducing factor,AIF)。采用变性条件下泛素化修饰实验检测FBXO22对AIF泛素化修饰的影响。通过短发夹RNA敲低FBXO22、过表达FBXO22检测AIF的表达水平。利用流式细胞仪检测FBXO22敲低或过表达对结肠癌细胞凋亡的影响。结果·免疫沉淀联合质谱分析显示FBXO22可与AIF结合,变性条件下泛素化修饰实验显示FBXO22可介导AIF的泛素化修饰。敲低FBXO22可上调AIF水平,过表达FBXO22可下调AIF水平。流式细胞术结果显示,敲低FBXO22可促进由甲基硝基亚硝基胍(N-methyl-N’-nitro-N-nitrosoguanidine,MNNG)等诱导的结肠癌细胞凋亡,过表达FBXO22则可抑制MNNG等诱导的结肠癌细胞凋亡。结论·FBXO22通过泛素化修饰降解AIF进而调控由AIF介导的结肠癌细胞凋亡。
基金the National Natural Science Foundation of China(Nos.32070584,81830028,31771398,82222016,and 8207040100)the Zhejiang Provincial Natural Science Foundation of China(No.LZ19C060001)the Fundamental Research Funds for the Central Universities(No.2019QNA6001)。
文摘Auditory neuropathy spectrum disorder(ANSD)represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function,but with the preservation of outer hair cell function.ANSD represents up to 15%of individuals with hearing impairments.Through mutation screening,bioinformatic analysis and expression studies,we have previously identified several apoptosis-inducing factor(AIF)mitochondria-associated 1(AIFM1)variants in ANSD families and in some other sporadic cases.Here,to elucidate the pathogenic mechanisms underlying each AIFM1 variant,we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system and constructed AIF-wild type(WT)and AIF-mutant(mut)(p.T260A,p.R422W,and p.R451Q)stable transfection cell lines.We then analyzed AIF structure,coenzyme-binding affinity,apoptosis,and other aspects.Results revealed that these variants resulted in impaired dimerization,compromising AIF function.The reduction reaction of AIF variants had proceeded slower than that of AIF-WT.The average levels of AIF dimerization in AIF variant cells were only 34.5%-49.7%of that of AIF-WT cells,resulting in caspase-independent apoptosis.The average percentage of apoptotic cells in the variants was 12.3%-17.9%,which was significantly higher than that(6.9%-7.4%)in controls.However,nicotinamide adenine dinucleotide(NADH)treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells.Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD,and introduce NADH as a potential drug for ANSD treatment.Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
