南京医科大学学报（自然科学版）2026，Vol.46Issue(9)：1315-1322,1330,9.DOI:10.7655/NYDXBNSN251472
Pin1负向调控Notch1信号通路抑制神经干细胞增殖
Pin1 negatively regulates the Notch1 signaling pathway to restrict the proliferation of neural stem cells
摘要
Abstract
Objective:To construct peptidyl-prolyl cis-trans isomerase never in mitosis A-interacting 1(Pin1)knockout C17.2 neural stem cell(NSC)lines using CRISPR/Cas9 technology,elucidate the impact of Pin1 deficiency on the proliferation capacity of embryonic NSCs,and explore the underlying molecular mechanisms based on transcriptome sequencing.Methods:Specific single guide RNAs(sgRNAs)targeting exon 2 of the mouse Pin1 gene were designed to construct the CRISPR/Cas9 targeting vectors.These vectors were transfected into C17.2 NSCs,followed by G418 selection to obtain Pin1 knockout single-cell colonies.The knockout efficiency and stemness maintenance(Nestin expression)were validated by immunofluorescence and Western blot analyses.Cell proliferation was evaluated using BrdU incorporation assays and Ki67 immunofluorescence staining.Differentially expressed genes(DEGs)were screened via RNA-Seq and subjected to pathway enrichment analysis.The expression of DEGs was further validated by quantitative real-time PCR(qRT-PCR)and Western blot.Finally,DAPT,a γ-secretase inhibitor,was employed to block the Notch signaling pathway to verify its role in Pin1-mediated cell proliferation.Results:Three C17.2 cell clones with biallelic Pin1 knockout were successfully generated.Compared to wild-type cells,Pin1 knockout cells maintained positive Nestin expression,but exhibited significantly increased BrdU incorporation and Ki67-positive rates(P＜0.05),indicating a state of hyperproliferation.RNA-Seq and subsequent validation experiments demonstrated that Pin1 deletion resulted in the significant activation of the Notch signaling pathway,characterized by the marked upregulation of the Notch1 protein and its downstream target gene Hes1(P＜0.05).Treatment with DAPT not only effectively inhibited the hyperactivation of the Notch1 pathway in Pin1 knockout cells but also significantly reduced their proliferation rate.Conclusion:Pin1 functions as a negative regulator in C17.2 NSCs,restricting excessive cellular proliferation by inhibiting the Notch1 signaling pathway.These findings reveal a novel mechanism by which Pin1 maintains proliferation homeostasis during the development of embryonic NSCs.关键词
Pin1/Notch1/CRISPR/Cas9/神经干细胞/增殖Key words
Pin1/Notch1/CRISPR/Cas9/neural stem cell/proliferation分类
医药卫生引用本文复制引用
潘婷,汪静,孙晓琦,陆文晗,李琳,戴一凡,王盈,杨海元..Pin1负向调控Notch1信号通路抑制神经干细胞增殖[J].南京医科大学学报（自然科学版）,2026,46(9):1315-1322,1330,9.基金项目
国家自然科学基金(81970164) （81970164）
