Objective:The purinergic receptor P2X2(P2RX2)encodes an ATP-gated ion channel permeable to Na+,K+,and especially Ca²⁺.Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafnes...Objective:The purinergic receptor P2X2(P2RX2)encodes an ATP-gated ion channel permeable to Na+,K+,and especially Ca²⁺.Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41(DFNA41),which manifests as high-frequency hearing loss,accelerated presbycusis,and increased susceptibility to noise-induced damage.Zebrafish,owing to their small size,rapid development,high fecundity,transparent embryos,and high gene conservation with humans,provide an ideal model for studying human diseases and developmental mechanisms.This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system,providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.Methods:Two CRISPR targets(sgRNA1 and sgRNA2)spaced 47 bp apart were designed within the zebrafish p2rx2 gene.Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen(TU)-strain zebrafish embryos.PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization(hpf).Surviving embryos were raised to adulthood(F0),tail-clipped,genotyped,and screened for positive mosaics.F1 heterozygotes were generated by outcrossing,and F2 homozygous mutants were obtained by intercrossing.Polymerase chain reaction(PCR)combined with sequencing verified mutation type and heritability.At 5 days post-fertilization(dpf),YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region.Auditory evoked potential(AEP)thresholds at 600,800,1000,and 2000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.Results:A stable p2rx2 knockout zebrafish line was successfully established.Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon(TAA),leading to early termination of protein translation and loss of function.Embryos developed normally with no gross malformations.At 5 dpf,mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type,although lateral line neuromasts were unaffected.AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type(all P<0.001),indicating reduced hearing sensitivity.Conclusion:We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology.p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies,indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception.The phenotype’s restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs.This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.展开更多
Objective:Middle ear cholesteatoma is a non-tumorous condition that typically leads to hearing loss,bone destruction,and other severe complications.Despite surgery being the primary treatment,the recurrence rate remai...Objective:Middle ear cholesteatoma is a non-tumorous condition that typically leads to hearing loss,bone destruction,and other severe complications.Despite surgery being the primary treatment,the recurrence rate remains high.Therefore,exploring the molecular mechanisms underlying cholesteatoma is crucial for discovering new therapeutic approaches.This study aims to explore the involvement of N6-methyladenosine(m^(6)A)methylation in long non-coding RNAs(lncRNAs)in the biological functions and related pathways of middle ear cholesteatoma.Methods:The m^(6)A modification patterns of lncRNA in middle ear cholesteatoma tissues(n=5)and normal post-auricular skin tissues(n=5)were analyzed using an lncRNA m^(6)A transcriptome microarray.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were conducted to identify potential biological functions and signaling pathways involved in the pathogenesis of middle ear cholesteatoma.Methylated RNA immunoprecipitation(MeRIP)-PCR was used to validate the m^(6)A modifications in cholesteatoma and normal skin tissues.Results:Compared with normal skin tissues,1525 lncRNAs were differentially methylated in middle ear cholesteatoma tissues,with 1048 showing hypermethylation and 477 showing hypomethylation[fold change(FC)≥3 or<1/3,P<0.05].GO enrichment analysis indicated that hypermethylated lncRNAs were involved in protein phosphatase inhibitor activity,neuron-neuron synapse,and regulation ofα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA)receptor activity.Hypomethylated lncRNAs were associated with mRNA methyltransferase activity,secretory granule membrane,and mRNA methylation.KEGG analysis revealed that hypermethylated lncRNAs were mainly associated with 5 pathways:the Hedgehog signaling pathway,viral protein interaction with cytokines and cytokine receptors,mitogen-activated protein kinase(MAPK)signaling pathway,cytokine-cytokine receptor interaction,and adrenergic signaling in cardiomyocytes.Hypomethylated lncRNAs were mainly involved in 4 pathways:Renal cell carcinoma,tumor necrosis factor signaling pathway,transcriptional misregulation in cancer,and cytokine-cytokine receptor interaction.Additionally,MeRIP-PCR confirmed the changes in m^(6)A methylation levels in NR_033339,NR_122111,NR_130744,and NR_026800,consistent with microarray analysis.Real-time PCR also confirmed the significant upregulation of MAPK1 and NF-κB,key genes in the MAPK signaling pathway.Conclusion:This study reveals the m^(6)A modification patterns of lncRNAs in middle ear cholesteatoma,suggests a direction for further research into the role of lncRNA m^(6)A modification in the etiology of cholesteatoma.The findings provide potential therapeutic targets for the treatment of middle ear cholesteatoma.展开更多
基金supported by the Natural Science Foundation of Hunan Province(2023JJ30753)the Innovative Construction Foundation of Hunan Province(2023SK4030)the Natural Science Foundation of Changsha(kq2208326),China。
文摘Objective:The purinergic receptor P2X2(P2RX2)encodes an ATP-gated ion channel permeable to Na+,K+,and especially Ca²⁺.Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41(DFNA41),which manifests as high-frequency hearing loss,accelerated presbycusis,and increased susceptibility to noise-induced damage.Zebrafish,owing to their small size,rapid development,high fecundity,transparent embryos,and high gene conservation with humans,provide an ideal model for studying human diseases and developmental mechanisms.This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system,providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.Methods:Two CRISPR targets(sgRNA1 and sgRNA2)spaced 47 bp apart were designed within the zebrafish p2rx2 gene.Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen(TU)-strain zebrafish embryos.PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization(hpf).Surviving embryos were raised to adulthood(F0),tail-clipped,genotyped,and screened for positive mosaics.F1 heterozygotes were generated by outcrossing,and F2 homozygous mutants were obtained by intercrossing.Polymerase chain reaction(PCR)combined with sequencing verified mutation type and heritability.At 5 days post-fertilization(dpf),YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region.Auditory evoked potential(AEP)thresholds at 600,800,1000,and 2000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.Results:A stable p2rx2 knockout zebrafish line was successfully established.Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon(TAA),leading to early termination of protein translation and loss of function.Embryos developed normally with no gross malformations.At 5 dpf,mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type,although lateral line neuromasts were unaffected.AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type(all P<0.001),indicating reduced hearing sensitivity.Conclusion:We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology.p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies,indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception.The phenotype’s restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs.This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
基金supported by the National Natural Science Foundation(82071036,82000973)the Natural Science Foundation of Hunan Province(2022JJ30821,2019JJ50967)the Special Project for the Construction of Innovative Provinces in Hunan Province(2023SK4030),China。
文摘Objective:Middle ear cholesteatoma is a non-tumorous condition that typically leads to hearing loss,bone destruction,and other severe complications.Despite surgery being the primary treatment,the recurrence rate remains high.Therefore,exploring the molecular mechanisms underlying cholesteatoma is crucial for discovering new therapeutic approaches.This study aims to explore the involvement of N6-methyladenosine(m^(6)A)methylation in long non-coding RNAs(lncRNAs)in the biological functions and related pathways of middle ear cholesteatoma.Methods:The m^(6)A modification patterns of lncRNA in middle ear cholesteatoma tissues(n=5)and normal post-auricular skin tissues(n=5)were analyzed using an lncRNA m^(6)A transcriptome microarray.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were conducted to identify potential biological functions and signaling pathways involved in the pathogenesis of middle ear cholesteatoma.Methylated RNA immunoprecipitation(MeRIP)-PCR was used to validate the m^(6)A modifications in cholesteatoma and normal skin tissues.Results:Compared with normal skin tissues,1525 lncRNAs were differentially methylated in middle ear cholesteatoma tissues,with 1048 showing hypermethylation and 477 showing hypomethylation[fold change(FC)≥3 or<1/3,P<0.05].GO enrichment analysis indicated that hypermethylated lncRNAs were involved in protein phosphatase inhibitor activity,neuron-neuron synapse,and regulation ofα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA)receptor activity.Hypomethylated lncRNAs were associated with mRNA methyltransferase activity,secretory granule membrane,and mRNA methylation.KEGG analysis revealed that hypermethylated lncRNAs were mainly associated with 5 pathways:the Hedgehog signaling pathway,viral protein interaction with cytokines and cytokine receptors,mitogen-activated protein kinase(MAPK)signaling pathway,cytokine-cytokine receptor interaction,and adrenergic signaling in cardiomyocytes.Hypomethylated lncRNAs were mainly involved in 4 pathways:Renal cell carcinoma,tumor necrosis factor signaling pathway,transcriptional misregulation in cancer,and cytokine-cytokine receptor interaction.Additionally,MeRIP-PCR confirmed the changes in m^(6)A methylation levels in NR_033339,NR_122111,NR_130744,and NR_026800,consistent with microarray analysis.Real-time PCR also confirmed the significant upregulation of MAPK1 and NF-κB,key genes in the MAPK signaling pathway.Conclusion:This study reveals the m^(6)A modification patterns of lncRNAs in middle ear cholesteatoma,suggests a direction for further research into the role of lncRNA m^(6)A modification in the etiology of cholesteatoma.The findings provide potential therapeutic targets for the treatment of middle ear cholesteatoma.
