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Title: [GINS1 Enhances Glycolysis, Proliferation and Metastasis in Lung Adenocarcinoma Cells by Activating the Notch/PI3K/AKT/mTORC1 Signaling Pathway].
Author: Huo Y, Xu X, Ma X, Feng Y.
Journal: Zhongguo Fei Ai Za Zhi; 2024 Oct 20; 27(10):735-744. PubMed ID: 39631830.
Abstract:
BACKGROUND: Lung cancer is the most common type of cancer, accounting for more than half of all cancer cases, with lung adenocarcinoma (LUAD) representing over half of lung cancer patients. Currently, the 5-year survival rate for metastatic LUAD patients remains low and there is an urgent need for new biomarkers as targets for targeted therapy. Go-Ichi-Ni-San 1 (GINS1), an important member of the GINS family, is closely related to the occurrence and development of human malignant tumors. This study aims to explore the role of GINS1 in glycolysis, proliferation, and metastasis of LUAD cells and the related molecular mechanisms. METHODS: The expression of GINS1 was analysed using bioinformatics between LUAD patients and healthy controls. The expression levels of GINS1 in LUAD and adjacent tissues were detected by immunohistochemistry and Western blot. Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the expression of GINS1 in LUAD cell lines A549, SK-LU-1, Calu-3, H1299 and BEAS-2B. Stably knockdown GINS1 in A549 cells and its negative control cell line, as well as stably overexpress GINS1 in H1299 cells and its negative control cell line, were constructed by lentiviral transduction. Colony formation test was used to detect cell proliferation. Scratch test was used to detect cell migration. Transwell test was used to detect cell invasion, and the test kits were used to detect glucose consumption and lactate production. The expression levels of glycolysis-related proteins, Notch signaling pathway proteins and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway proteins were detected by Western blot. The Notch receptor agonist Jagged1 was added to cells from the shGINS1-A549 group and the Notch receptor inhibitor LY3039478 was added to cells from the GINS1-OE-H1299 group for the regression assay. RESULTS: The expression of GINS1 was up-regulated in LUAD patients, tissues and cell lines, and correlated with overall survival (P<0.05). Knockdown of GINS1 significantly inhibited the proliferation, migration and invasion of A549 cells (P<0.05), while overexpression of GINS1 significantly enhanced the proliferation, migration and invasion of H1299 cells (P<0.05). Furthermore, knockdown of GINS1 resulted in reduced glucose consumption, reduced lactate production, and reduced expression levels of glycolytic-related proteins in A549 cells (P<0.05); overexpression of GINS1 enhanced glycolytic level in H1299 cells (P<0.05). The expression levels of Notch1, Notch3, phosphorylated-PI3K (p-PI3K), phosphorylated-AKT (p-AKT) and phosphorylated-mTORC1 (Ser2448)[p-mTORC1 (Ser2448)] in A549 cells were significantly decreased by GINS1 knockdown (P<0.05), while the expression levels of PI3K, AKT, mTOR and p-mTORC2 (Ser2481) were not significantly changed (P>0.05). Overexpression of GINS1 increased the levels of Notch1, Notch3 and PI3K/AKT/mTORC1 pathway phosphorylated proteins in H1299 cells (P<0.05). Jagged1 significantly reversed the inhibition of glycolysis, proliferation and metastasis induced by GINS1 knockdown in A549 cells (P<0.05), and LY3039478 significantly inhibited the enhancement of glycolysis, proliferation and metastasis induced by GINS1 overexpression in H1299 cells (P<0.05). CONCLUSIONS: The expression of GINS1 enhances the expression of Notch1 and Notch3 receptors, and then phosphorylates and activates the downstream PI3K/AKT/mTORC1 signaling pathway to enhance the glycolysis, proliferation and metastasis of LUAD cells. 【中文题目：GINS1通过激活Notch/PI3K/AKT/mTORC1 信号通路增强肺腺癌细胞糖酵解、增殖和转移】【中文摘要：背景与目的肺癌是所有癌症类型中占比最大的一种，其中肺腺癌（lung adenocarcinoma, LUAD）占肺癌患者的一半以上。目前，转移性LUAD患者5年生存率仍然较低，迫切需要新型生物标志物作为靶向治疗的靶点。Go-Ichi-Ni-San1（GINS1）是GINS家族的重要成员，与人类恶性肿瘤的发生和发展密切相关。本研究旨在探究GINS1在LUAD细胞糖酵解、增殖和转移过程中的作用及相关分子机制。方法通过生物信息学分析GINS1在LUAD患者和健康人群中的表达差异。通过免疫组织化学染色和Western blot检测GINS1在LUAD组织和癌旁组织中的表达水平，采用Western blot和实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction, qRT-PCR）检测GINS1在LUAD细胞系A549、SK-LU-1、Calu-3、H1299和人正常支气管上皮细胞系BEAS-2B中的表达水平。通过慢病毒转染细胞的方法构建稳定敲低GINS1的A549细胞株（shGINS1-A549）及其阴性对照细胞株（shGINS1-NC-A549）、稳定过表达GINS1的H1299细胞株（GINS1-OE-H1299）及其阴性对照细胞株（GINS1-OENC-H1299）。通过集落形成试验检测细胞增殖能力，划痕试验检测细胞迁移能力，Transwell试验检测细胞侵袭能力，试剂盒检测细胞对葡萄糖的消耗量以及乳酸的产量，Western blot检测糖酵解相关蛋白、Notch信号通路蛋白和磷脂酰肌醇-3-激酶（phosphatidylinositol-3-kinase, PI3K）/蛋白激酶B（protein kinase B, AKT）和哺乳动物雷帕霉素蛋白（mammalian target of rapamycin, mTOR）信号通路蛋白表达水平。向shGINS1-A549组细胞加入Notch受体激动剂Jagged1，向GINS1-OE-H1299组细胞加入Notch受体抑制剂LY3039478，进行回复试验。结果 GINS1在LUAD患者、组织和细胞系中均表达上调，并与患者总生存期有关（P<0.05）。敲低GINS1后，A549细胞的增殖、迁移和侵袭能力均受到显著抑制（P<0.05）；过表达GINS1则显著增强了H1299细胞的增殖、迁移和侵袭能力（P<0.05）。此外，敲低GINS1导致A549细胞对葡萄糖的消耗量减少，乳酸产量减少，糖酵解相关蛋白表达水平降低（P<0.05）；过表达GINS1则增强了H1299细胞糖酵解水平（P<0.05）。GINS1敲低导致A549细胞Notch1、Notch3、p-PI3K、p-AKT和p-mTORC1（Ser2448）蛋白表达水平显著降低（P<0.05），而PI3K、AKT、mTOR、p-mTORC2（Ser2481）蛋白表达水平无显著变化（P>0.05）；GINS1过表达则升高了H1299细胞Notch1、Notch3、PI3K/AKT/mTORC1通路磷酸化蛋白水平（P<0.05）。Jagged1显著逆转了由于GINS1敲低导致的A549细胞糖酵解、增殖和转移活性抑制（P<0.05）；LY3039478显著抑制了由于GINS1过表达诱导的H1299细胞糖酵解、增殖和转移活性增强（P<0.05）。结论 GINS1的表达通过促进Notch1、Notch3受体表达水平升高，进而磷酸化激活下游PI3K/AKT/mTORC1信号通路，增强LUAD细胞糖酵解、增殖和转移。 】【中文关键词：肺腺癌；GINS1； Notch；糖酵解；增殖；转移】.

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