乳腺癌中心体调控蛋白SEC23B在肿瘤浸润转移中的作用及其机制研究

doi:10.19401/j.cnki.1007-3639.2023.02.008

摘要/Abstract

摘要：

背景与目的： 新的中心体调控蛋白Sec23同系物B（Sec23 homolog B，SEC23B）可以调节细胞中的自噬，从而提供有助于肿瘤生长和远处转移的能量和营养。然而，这种作用是否参与乳腺癌的浸润转移尚不清楚。本研究旨在探讨SEC23B在乳腺癌浸润转移中的作用及其分子机制。方法： 使用Kaplan-Meier Plotter数据库和HCMDB数据库分析SEC23B表达与乳腺癌预后、转移之间的关联。将MCF-7细胞分为载体组（control）和SEC23B过表达质粒组（SEC23B），以及将MDA231-LM2细胞分为SEC23B敲低组（sh-SEC23B）和对照组（sh-NC）。采用蛋白质印迹法（Western blot）检测乳腺癌细胞中SEC23B、自噬相关蛋白[死骨片1（SQSTM1，p62）、微管相关蛋白1的轻链3（microtubule-associated-protein light-chain-3，LC3）]和细胞外调节蛋白激酶（extracellular-regulated protein kinases，ERK）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）信号转导通路蛋白的表达。通过transwell实验和伤口愈合实验评估细胞迁移，采用免疫荧光染色检测细胞中自噬颗粒。此外，构建体内腹膜内肿瘤模型研究SEC23B体内对乳腺癌细胞转移的影响。结果： SEC23B高表达的乳腺癌患者的总生存期明显短于SEC23B低表达的患者。SEC23B在转移性乳腺癌中的表达明显高于没有转移的乳腺癌原发性乳腺癌细胞（MCF-7、BT-549、MDA-MB-468和MDA-MB-453）中的SEC23B蛋白水平低于转移性乳腺癌细胞（MDA231-LM2和ZR-75-30）。与control组相比，SEC23B组明显加速了细胞迁移（P<0.001）。与sh-NC组相比，sh-SEC23B组明显降低了细胞迁移（P<0.001）。与control组相比，SEC23B组LC3A/B的表达水平和自噬颗粒的形成明显增加，而p62蛋白表达和mTOR、ERK的磷酸化水平降低。与sh-NC组相比，sh-SEC23B组LC3A/B表达水平和自噬颗粒的形成显著降低，和mTOR、ERK的磷酸化水平升高，特别是在饥饿条件下结果更显著。在体内实验中，与sh-NC组相比，sh-SEC23B组肿瘤重量显著降低（P<0.01），并且小鼠肿瘤组织中坏死组织增多和肺组织肿瘤转移减少。sh-SEC23B组小鼠肺转移肿瘤数和LC3免疫组织化学染色明显低于sh-NC组（P<0.01）。结论： SEC23B通过抑制ERK/mTOR信号转导通路来诱导乳腺癌自噬，并促进癌细胞转移。

关键词: Sec23同系物B, 乳腺癌, 自噬, 转移, ERK/mTOR信号转导通路

Abstract:

Background and purpose: The novel centrosomal regulatory protein Sec23 homolog B (SEC23B) regulates autophagy in cells, thereby providing energy and nutrients that contribute to tumor growth and distant metastasis. However, whether this role is involved in the mechanism of infiltrative metastasis in breast cancer is unclear. The aim of this study was to investigate the molecular mechanism by which SEC23B promotes breast cancer infiltration and metastasis. Methods: The association between SEC23B expression and breast cancer prognosis and metastasis was analyzed using Kaplan-Meier Plotter database and HCMDB database. MCF-7 cells were divided into vector group (control) and SEC23B overexpression plasmid group (SEC23B), and MDA231-LM2 cells were divided into SEC23B knockdown group (sh-SEC23B) and control group (sh-NC). Western blot was used to detect the expressions of SEC23B, autophagy-associated proteins [dead bone fragment 1 (SQSTM1, p62), microtubule-associated-protein light-chain-3 (LC3)] and extracellular-regulated protein kinases (ERK)/mammalian target of rapamycin (mTOR) pathway proteins in breast cancer cells. Cell migration was assessed by transwell assay and wound healing assay, and autophagic granules in cells were detected by immunofluorescence staining. In addition, an in vivo intraperitoneal tumor model was constructed to study the effect of SEC23B on breast cancer cell metastasis in vivo. Results: The overall survival of breast cancer patients with high SEC23B expression was significantly shorter than that of patients with low SEC23B expression. The expression of SEC23B in metastatic breast cancer was significantly higher than that in breast cancer without metastasis. SEC23B protein levels were lower in primary breast cancer cells (MCF-7, BT-549, MDA-MB-468 and MDA-MB-453) than in metastatic breast cancer cells (MDA231-LM2 and ZR-75-30). Compared with the control group, the SEC23B group significantly accelerated cell migration (P<0.001). Compared with the sh-NC group, the sh-SEC23B group significantly reduced cell migration (P<0.001). Compared with the control group, the expression level of LC3A/B and the formation of autophagy particles in the SEC23B group were significantly increased, while the expression of p62 protein and the phosphorylation levels of mTOR and ERK were decreased. Compared with the sh-NC group, the LC3A/B expression level and the formation of autophagy particles were significantly decreased in the sh-SEC23B group, and the phosphorylation levels of mTOR and ERK were increased, especially under starvation conditions. In in vivo experiments, tumor weight was significantly lower in the sh-SEC23B group compared with the sh-NC group (P<0.01), and there was an increase in necrotic tissue and a decrease in tumor metastasis in lung tissue in mice. sh-SEC23B group mice had a significantly lower number of lung metastatic tumors and LC3 immunohistochemical staining than the sh-NC group (P<0.01). Conclusions: SEC23B induces breast cancer autophagy and promotes cancer cell metastasis by inhibiting ERK/mTOR signaling pathway.

Key words: Sec23 homolog B, Breast cancer, Autophagy, Metastasis, ERK/mTOR signaling pathway

中图分类号:

R737.9

冷婕, 邱国春, 张菠, 蒲艳. 乳腺癌中心体调控蛋白SEC23B在肿瘤浸润转移中的作用及其机制研究[J]. 中国癌症杂志, 2023, 33(2): 152-161.

LENG Jie, QIU Guochun, ZHANG Bo, PU Yan. Mechanism of breast cancer centrosome regulatory protein SEC23B on tumor invasion and metastasis[J]. China Oncology, 2023, 33(2): 152-161.

图/表 5

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