OBJECTIVES: 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, 1 000, and 2 000 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. CONCLUSIONS 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.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

Objective : To clarify the targeted regulatory relationship between long non-coding RNA ( lncRNAs) lnc-CCDC117-1 and oncogenic transcription factor c-Myc，and to explore the effect of lnc-CCDC117-1 knockdown and overexpression on the development of tongue squamous cell carcinoma cells． Methods : Lentiviral vectors car- rying flag-c-Myc，plko．1-shc-Myc and their controls were transfected into HN6，SCC9 and CAL27 cells respective- ly，and real-time quantitative PCR(qRT-PCR) was used to detect the expression of lnc-CCDC117-1．In addition， the intracellular localization of lnc-CCDC117-1 was detected by fluorescence in situ hybridization．On this basis，the lnc-CCDC117-1 expression vector and knockdown vector were constructed and transfected with HN6 ，SCC9， CAL27 cells，CCK-8 method，cloning and formation to verify the proliferation of tongue squamous cell. Results: LncRNAs positively regulated by c-Myc were initially screened by gene chip technology．After overexpression or c- Myc knockdown in tongue squamous cell HN6，these lncRNAs were verified to be consistent with the results of the chip，and the expression difference of lnc-CCDC117-1 was found to be the most significant．qRT-PCR test showed that c-Myc had a positive regulatory effect on lnc-CCDC117-1．When c-Myc was overexpressed，the expression of lnc-CCDC117-1 could be significantly up-regulated．lnc-CCDC117-1 expression was significantly down-regulated by c-Myc knockdown．Dual luciferase reporter genes showed that c-Myc could target the regulation of lnc-CCDC117-1， and c-Myc was involved in regulating and enhancing the transcriptional activity of lnc-CCDC117-1．lnc-CCDC117- 1 mainly existed in the nucleus of the cell．qRT-PCR results showed that the expression of lnc-CCDC117-1 and c- Myc significantly decreased by sh-lnc-CCDC117-1．The expression of lnc-CCDC117-1 was significantly up-regula- ted by + lnc-CCDC117-1．The results of growth curve assay，CCK-8 assay，cell scratch assay and cloning forma- tion showed that overexpression of lnc-CCDC117-1 significantly promoted the proliferation and migration of tongue squamous cell cells，while knockdown of lnc-CCDC117-1 could inhibit the growth and proliferation of tongue squa- mous cell carcinoma cells． Conclusion lnc-CCDC117-1 is positively regulated by c-Myc，and overexpression of lnc-CCDC117-1 can promote cell proliferation，on the contrary，inhibit cells growth.