Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-asso...Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.展开更多
目的:探讨长链非编码RNA TFAP2A-AS1对子宫内膜癌细胞增殖、侵袭和迁移的影响及机制。方法:选择50例子宫内膜癌组织和对应的癌旁组织。选取子宫内膜癌细胞株(RL95-2、HEC-1A、HHUA、HEC-1B及Ishikawa),子宫内膜上皮细胞hEEC。子宫内膜...目的:探讨长链非编码RNA TFAP2A-AS1对子宫内膜癌细胞增殖、侵袭和迁移的影响及机制。方法:选择50例子宫内膜癌组织和对应的癌旁组织。选取子宫内膜癌细胞株(RL95-2、HEC-1A、HHUA、HEC-1B及Ishikawa),子宫内膜上皮细胞hEEC。子宫内膜癌细胞株Ishikawa转染si-TFAP2A-AS1(si-TFAP2A-AS1组)、si-NC(si-NC组)、miR-9-5p mimics(miR-9-5p组)及miR-NC(miR-NC组);RL95-2细胞转染OE-TFAP2A-AS1(TFAP2A-AS1组)、Vector(Vector组)、sh-miR-9-5p(sh-miR-9-5p组)及sh-NC(sh-NC组)。CCK-8检测细胞增殖能力,Transwell检测细胞侵袭、迁移能力,双荧光素酶实验、pull down实验分析TFAP2A-AS1与miR-9-5p的靶向关系;miR-9-5p与ERK的靶向关系,实时荧光定量PCR(RT-qPCR)检测子宫内膜癌组织、癌旁组织、子宫内膜癌细胞及hEEC细胞TFAP2AAS1、miR-9-5p表达水平,Western blot检测细胞外调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)水平。结果:子宫内膜癌组织中TFAP2A-AS1表达水平低于癌旁组织,miR-9-5p表达水平高于癌旁组织(P<0.05)。子宫内膜癌组织TFAP2A-AS1、miR-9-5p表达水平与分化程度、肿瘤直径、TNM分期及淋巴结转移有关。子宫内膜癌组织中TFAP2A-AS1、miR-9-5p表达水平呈负相关(r=-0.782,P=0.002)。与hEEC细胞比较,RL95-2、HEC-1A、HHUA、HEC-1B及Ishikawa细胞TFAP2A-AS1表达水平降低,miR-9-5p表达水平升高(P<0.05)。与si-NC组比较,si-TFAP2A-AS1组细胞48 h、72 h OD值,侵袭、迁移细胞数均升高(P<0.05);与Vector组比较,TFAP2A-AS1组细胞48 h、72 h OD值,侵袭、迁移细胞数均降低(P<0.05)。与miR-NC组比较,miR-9-5p组细胞48 h、72 h OD值,侵袭、迁移细胞数均升高(P<0.05);与sh-NC组比较,sh-miR-9-5p组细胞48 h、72 h OD值,侵袭、迁移细胞数均降低(P<0.05)。TFAP2A-AS1负性调控miR-9-5p、p-ERK表达水平,miR-9-5p可靶向调控ERK蛋白。结论:过表达TFAP2A-AS1通过靶向下调miR-9-5p,抑制ERK通路,最终抑制子宫内膜癌细胞恶性生物学行为。展开更多
基金supported by the National Natural Science Foundation of China,Nos.82071008(to BL)and 82004001(to XJ)Medical Science and Technology Program of Health Commission of Henan Province,No.LHGJ20210072(to RQ)Science and Technology Department of Henan Province,No.212102310307(to XJ)。
文摘Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.
文摘目的:探讨长链非编码RNA TFAP2A-AS1对子宫内膜癌细胞增殖、侵袭和迁移的影响及机制。方法:选择50例子宫内膜癌组织和对应的癌旁组织。选取子宫内膜癌细胞株(RL95-2、HEC-1A、HHUA、HEC-1B及Ishikawa),子宫内膜上皮细胞hEEC。子宫内膜癌细胞株Ishikawa转染si-TFAP2A-AS1(si-TFAP2A-AS1组)、si-NC(si-NC组)、miR-9-5p mimics(miR-9-5p组)及miR-NC(miR-NC组);RL95-2细胞转染OE-TFAP2A-AS1(TFAP2A-AS1组)、Vector(Vector组)、sh-miR-9-5p(sh-miR-9-5p组)及sh-NC(sh-NC组)。CCK-8检测细胞增殖能力,Transwell检测细胞侵袭、迁移能力,双荧光素酶实验、pull down实验分析TFAP2A-AS1与miR-9-5p的靶向关系;miR-9-5p与ERK的靶向关系,实时荧光定量PCR(RT-qPCR)检测子宫内膜癌组织、癌旁组织、子宫内膜癌细胞及hEEC细胞TFAP2AAS1、miR-9-5p表达水平,Western blot检测细胞外调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)水平。结果:子宫内膜癌组织中TFAP2A-AS1表达水平低于癌旁组织,miR-9-5p表达水平高于癌旁组织(P<0.05)。子宫内膜癌组织TFAP2A-AS1、miR-9-5p表达水平与分化程度、肿瘤直径、TNM分期及淋巴结转移有关。子宫内膜癌组织中TFAP2A-AS1、miR-9-5p表达水平呈负相关(r=-0.782,P=0.002)。与hEEC细胞比较,RL95-2、HEC-1A、HHUA、HEC-1B及Ishikawa细胞TFAP2A-AS1表达水平降低,miR-9-5p表达水平升高(P<0.05)。与si-NC组比较,si-TFAP2A-AS1组细胞48 h、72 h OD值,侵袭、迁移细胞数均升高(P<0.05);与Vector组比较,TFAP2A-AS1组细胞48 h、72 h OD值,侵袭、迁移细胞数均降低(P<0.05)。与miR-NC组比较,miR-9-5p组细胞48 h、72 h OD值,侵袭、迁移细胞数均升高(P<0.05);与sh-NC组比较,sh-miR-9-5p组细胞48 h、72 h OD值,侵袭、迁移细胞数均降低(P<0.05)。TFAP2A-AS1负性调控miR-9-5p、p-ERK表达水平,miR-9-5p可靶向调控ERK蛋白。结论:过表达TFAP2A-AS1通过靶向下调miR-9-5p,抑制ERK通路,最终抑制子宫内膜癌细胞恶性生物学行为。
