PIN1 在人胶质母细胞瘤模型中端粒维持和致癌行为中的关键作用。

Key role of PIN1 in telomere maintenance and oncogenic behavior in a human glioblastoma model.

机构信息

Molecular Oncology Unit, Center of Molecular and Translational Oncology, Department of Science and Technology, National University of Quilmes, Bernal, Buenos Aires B1876BXD, Argentina.

Antitumor Drug Evaluation and Development Unit, Center of Molecular and Translational Oncology, Department of Science and Technology, National University of Quilmes, Bernal, Buenos Aires B1876BXD, Argentina.

出版信息

Oncol Rep. 2023 May;49(5). doi: 10.3892/or.2023.8528. Epub 2023 Mar 24.

DOI:10.3892/or.2023.8528

PMID:36960859

Abstract

PIN1 is the only known enzyme capable of recognizing and isomerizing the phosphorylated Serine/Threonine‑Proline motif. Through this mechanism, PIN1 controls diverse cellular functions, including telomere maintenance. Both PIN1 overexpression and its involvement in oncogenic pathways are involved in several cancer types, including glioblastoma (GBM), a lethal disease with poor therapeutic resources. However, knowledge of the role of PIN1 in GBM is limited. Thus, the present work aimed to study the role of PIN1 as a telomere/telomerase regulator and its contribution to tumor biology. PIN1 knockout (KO) LN‑229 cell variant using CRISPR/Cas9 was developed and compared with PIN1 LN‑229 expressing cells. To study the effect of PIN1 absence, status of NF‑κB pathway was evaluated by luciferase reporter gene assay and quantitative PCR. Results revealed that PIN1 deletion in GBM cells diminished the active levels of NF‑κB and decrease the transcription of il‑8 and htert genes. Then, telomere/telomerase related processes were studied by RQ‑TRAP assay and telomere length determination by qPCR, obtaining a reduction both in telomerase activity as in telomere length in PIN1 KO cells. In addition, measurement of SA β‑galactosidase and caspase‑3 activities revealed that loss of PIN1 triggers senescence and apoptosis. Finally, migration, cell cycle progression and tumorigenicity were studied by flow cytometry/western blot, Transwell assay and experiments, respectively. PIN1 deletion decreased migration as well as cell cycle progression by increasing doubling time and also resulted in the loss of LN‑229 cell ability to form tumors in mice. These results highlight the role of PIN1 in telomere homeostasis and GBM progression, which supports PIN1 as a potential molecular target for the development of novel therapeutic agents for GBM treatment.

摘要

PIN1 是唯一已知能够识别并使磷酸化丝氨酸/苏氨酸-脯氨酸基序发生异构化的酶。通过这种机制，PIN1 控制着多种细胞功能，包括端粒维持。PIN1 的过表达及其参与致癌途径与多种癌症类型有关，包括胶质母细胞瘤 (GBM)，这是一种治疗资源有限的致命疾病。然而，PIN1 在 GBM 中的作用知之甚少。因此，本工作旨在研究 PIN1 作为端粒/端粒酶调节剂的作用及其对肿瘤生物学的贡献。利用 CRISPR/Cas9 开发了 PIN1 敲除 (KO) LN-229 细胞变体，并将其与表达 PIN1 的 LN-229 细胞进行了比较。为了研究 PIN1 缺失的影响，通过荧光素酶报告基因检测和定量 PCR 评估了 NF-κB 通路的状态。结果表明，GBM 细胞中 PIN1 的缺失降低了 NF-κB 的活性水平，并降低了 il-8 和 htert 基因的转录。然后，通过 RQ-TRAP 测定和 qPCR 测定端粒长度来研究端粒/端粒酶相关过程，在 PIN1 KO 细胞中观察到端粒酶活性和端粒长度均降低。此外，通过测定 SA β-半乳糖苷酶和 caspase-3 活性，发现 PIN1 的缺失会触发衰老和细胞凋亡。最后，通过流式细胞术/蛋白质印迹、Transwell 测定和实验分别研究了迁移、细胞周期进程和致瘤性。PIN1 的缺失通过增加倍增时间来减少迁移和细胞周期进程，并导致 LN-229 细胞丧失在小鼠中形成肿瘤的能力。这些结果突出了 PIN1 在端粒动态平衡和 GBM 进展中的作用，支持 PIN1 作为开发用于 GBM 治疗的新型治疗剂的潜在分子靶标。