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

目的: 利用CRISPR/Cas9技术构建肽基脯氨酰顺反式异构酶永不分裂基因A相互作用蛋白（peptidyl-prolyl cis-trans isomerase never in mitosis A-interacting 1， Pin1）基因敲除的C17.2神经干细胞，揭示Pin1缺失对胚胎神经干细胞增殖能力的影响，并基于转录组测序探究其潜在的分子机制。方法: 针对小鼠 Pin1外显子2设计特异性小向导RNA（single guide RNA，sgRNA），构建靶向 Pin1的CRISPR/Cas9打靶载体，转染C17.2神经干细胞并经G418筛选鉴定获得 Pin1敲除的单细胞克隆。利用免疫荧光染色和Western blot验证 Pin1敲除效率及干性标志物Nestin的表达；通过BrdU掺入实验和Ki67免疫荧光染色评估细胞增殖能力的改变。通过RNA-Seq筛选差异表达基因并进行通路富集分析，通过qRT-PCR和Western blot验证差异基因的表达；最后利用γ-分泌酶抑制剂DAPT阻断Notch通路，验证该通路在Pin1调控细胞增殖中的作用。结果: 成功获得3个 Pin1双等位基因敲除的C17.2细胞克隆。与野生型细胞相比， Pin1敲除细胞维持Nestin阳性表达，BrdU掺入率和Ki67阳性率显著升高（P<0.05），呈过度增殖状态。RNA-Seq及验证实验表明，Pin1缺失导致Notch信号通路显著激活，表现为Notch1受体蛋白及其下游靶基因 Hes1表达水平显著上调（P<0.05）。DAPT处理不仅有效抑制了 Pin1敲除细胞中Notch1通路的过度激活，而且显著降低了细胞的增殖速率。结论: Pin1在C17.2神经干细胞中作为一种负向调控因子，通过抑制Notch1信号通路来限制细胞的过度增殖。这一发现揭示了Pin1在胚胎神经干细胞发育阶段维持增殖稳态的新机制。

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

参考文献

[1] YAFFE M B, RITTINGER K, VOLINIA S, et al. The structural basis for 14—3—3: phosphopeptide binding specificity[J]. Cell, 1997, 91(7):961-971.

[2] FAGIANI F, GOVONI S, RACCHI M, et al. The peptidyl—prolyl isomerase Pin1 in neuronal signaling: from neurodevelopment to neurodegeneration[J]. Mol Neurobiol, 2021, 58(3):1062-1073.

[3] HLEIHEL R, EL HAJJ H, WU H C, et al. A Pin1/PML/P53 axis activated by retinoic acid in NPM—1c acute myeloid leukemia[J]. Haematologica, 2021, 106(12):3090-3099.

[4] LANNI C, MASII M, RACCHI M, et al. Cancer and Alzheimer's disease inverse relationship: an age—associated diverging derailment of shared pathways[J]. Mol Psychiatry, 2021, 26(1):280-295.

[5] QIU C X, LI Z X, LEIGH D A, et al. The role of the Pin1—cis p—tau axis in the development and treatment of vascular contribution to cognitive impairment and dementia and preeclampsia[J]. Front Cell Dev Biol, 2024, 12:1343962.

[6] CALIGIURI I, VINCENZO C, ASANO T, et al. The metabolic crosstalk between PIN1 and the tumour microenvironment[J]. Semin Cancer Biol, 2023, 91:143-157.

[7] WANG X, LEE D, XU H, et al. PIN1 prolyl isomerase promotes initiation and progression of bladder cancer through the SREBP2—mediated cholesterol biosynthesis pathway[J]. Cancer Discov, 2025, 15(3):633-655.

[8] XIE G, OKUDA S, GAO J Y, et al. The central role of Pin1 in age—related cancer signaling pathways[J]. Semin Cancer Biol, 2025, 114:173-194.

[9] YU J H, IM C Y, MIN S H. Function of PIN1 in cancer development and its inhibitors as cancer therapeutics[J]. Front Cell Dev Biol, 2020, 8: 120.

[10] BALASTIK M, ZHOU X Z, ALBERICH—JORDA M, et al. Prolyl isomerase Pin1 regulates axon guidance by stabilizing CRMP2A selectively in distal axons[J]. Cell Rep, 2015, 13(4):812-828.

[11] MALTER J S. Pin1 and Alzheimer's disease[J]. Transl Res, 2023, 254:24-33.

[12] ZHENG F, LI Y, ZHANG F, et al. Cobalt induces neurodegenerative damages through Pin1 inactivation in mice and human neuroglioma cells[J]. J Hazard Mater, 2021, 419:126378.

[13] SHABANI K, PIGEON J, BENAISSA T Z M, et al. The temporal balance between self—renewal and differentiation of human neural stem cells requires the amyloid precursor protein[J]. Sci Adv, 2023, 9(24):eadd5002.

[14] SONG P W, HAN T Y, WU Z M, et al. Transplantation of neural stem cells loaded in an IGF—1 bioactive supramolecular nanofiber hydrogel for the effective treatment of spinal cord injury[J]. Adv Sci, 2024, 11(17):e2306577.

[15] HAYASHI Y, LIN H T, LEE C C, et al. Effects of neural stem cell transplantation in Alzheimer's disease models[J]. J Biomed Sci, 2020, 27(1):29.

[16] LIU Y F, LIU H T, CHANG C, et al. Stereotactically intracerebral transplantation of neural stem cells for ischemic stroke attenuated inflammatory responses and promoted neurogenesis: an experimental study with monkeys[J]. Int J Surg, 2024, 110(9):5417-5433.

[17] RAO J, LI H, ZHANG H, et al. Periplaneta Americana (L.) extract activates the ERK/CREB/BDNF pathway to promote post—stroke neuroregeneration and recovery of neurological functions in rats[J]. J Ethnopharmacol, 2024, 321:117400.

[18] SNYDER E Y, DEITCHER D L, WALSH C, et al. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum[J]. Cell, 1992, 68(1):33-51.

[19] STALLINGS N R, O'NEAL M A, HU J, et al. Long—term normalization of calcineurin activity in model mice rescues Pin1 and attenuates Alzheimer's phenotypes without blocking peripheral T cell IL—2 response[J]. Alzheimers Res Ther, 2023, 15(1):179.

[20] WANG S W, GAO C, ZHENG Y M, et al. Current applications and future perspective of CRISPR/Cas9 gene editing in cancer[J]. Mol Cancer, 2022, 21(1):57.

[21] FRANGOUL H, ALTSHULER D, CAPPELLINI M D, et al. CRISPR—Cas9 gene editing for sickle cell disease and β—thalassemia[J]. N Engl J Med, 2021, 384(3):252-260.

[22] RUSTIGHI A, ZANNINI A, CAMPANER E, et al. PIN1 in breast development and cancer: a clinical perspective[J]. Cell Death Differ, 2017, 24(2):200-211.

[23] LA R S. Diagnostic, prognostic, and predictive role of Ki67 proliferative index in neuroendocrine and endocrine neoplasms: past, present, and future[J]. Endocr Pathol, 2023, 34(1):79-97.

[24] HUANG X, WANG Y J, LI Q S, et al. Coupling of stemness maintenance with cell cycle control in stem cells[J]. Front Cell Dev Biol, 2025, 13:1693489.

[25] HAARWOOD D S L, PEDERSEN V, BAGER N S, et al. Glioblastoma cells increase expression of notch signaling and synaptic genes within infiltrated brain tissue[J]. Nat Commun, 2024, 15(1):7857.

基金资助

国家自然科学基金(81970164)