过表达RGL1通过激活CDC42/RAC1复合体上调运动型黏着斑组装促进结直肠癌转移

doi:10.12122/j.issn.1673-4254.2025.05.16

摘要/Abstract

摘要：

目的探究Ral鸟嘌呤核苷酸解离激活样因子1（RGL1）在转移性结直肠癌中的作用及机制。方法分析GEO数据库中RGL1在结直肠癌转移性和非转移性中的表达差异。收集珠江医院2020年1月~2022年12月50例临床转移性与非转移性结直肠癌患者，采用qPCR和免疫组化分析RGL1与结直肠癌转移的关系。体外构建稳定过表达和稳定干扰RGL1基因的细胞株，分别分组为：过表达对照组（veh）与过表达组（RGL1+），敲低对照组（shNC）与敲低组（shRGL1）。Western blotting实验验证过表达和干扰效率。Transwell实验体外验证细胞表型，明确RGL1对肿瘤细胞侵袭、迁移能力的影响。体内实验采用小鼠盲肠原位种植肝转移模型验证动物表型，明确RGL1对肿瘤转移的影响。细胞-基质黏附检测实验、免疫荧光实验验证RGL1与黏着斑形成的关系，免疫共沉淀实验探究RGL1与GTP酶活化的关系。结果与非转移性结直肠癌相比，RGL1在转移性结直肠癌中高表达（P<0.05）。与对照组相比，过表达RGL1细胞中该基因上调（P<0.05），细胞迁移、侵袭能力增强（P<0.05），小鼠出现结直肠癌肝转移；并且加速了结直肠癌细胞运动型黏着斑的组装（P<0.05），促进了运动型黏着斑的形成，上调CDC42/RAC1的活化态与RGL1的结合。结论 RGL1在转移性结直肠癌中高表达，通过激活CDC42/RAC1复合体上调运动型黏着斑组装，从而促进结直肠癌转移。RGL1有望成为防治结直肠癌转移的新靶点。

关键词: RGL1, 转移性结直肠癌, 运动型黏着斑, CDC42, RAC1

Abstract:

Objective To investigate the regulatory role of Ral guanine nucleotide dissociation stimulator-like 1 (RGL1) in metastasis of colorectal cancer (CRC). Methods We analyzed the differential expression of RGL1 between metastatic and non-metastatic CRC in GEO database, and examined its expression in 25 patients with metastatic CRC and 25 patients with non-metastatic CRC treated in Zhujiang Hospital between January, 2020 and December, 2022 using quantitative PCR (qPCR) and immunohistochemistry. HCT116 cell lines with stable RGL1 overexpression and SW480 cells with RGL1 knockdown were established using lentiviral vecors, and the changes in invasion and migration abilities of the cells were assessed using Transwell invasion and migration assays. The transduced cells were injected into the serosa of the cecum of nude mice, and tumor growth and liver metastasis were observed 8 weeks later. Fibronectin adhesion assays and immunofluorescence experiments were employed to assess the relationship between RGL1 and focal adhesion formation, and co-immuno-precipitation assays were performed to explore the interaction between RGL1 and GTPase activation. Results Compared with non-metastatic CRC, metastatic CRC showed significantly upregulated expression of RGL1. HCT116 cells overexpressing RGL1 exhibited obviously enhanced migration and invasion in vitro with increased capacity for liver metastasis in nude mice. RGL1 overexpression strongly accelerated focal adhesion assembly, facilitated the formation of motile focal adhesions, and enhanced the binding of activated CDC42/RAC1 complex to RGL1. Conclusion RGL1 is highly expressed in metastatic CRC and promotes distant metastasis of CRC by activating the CDC42/RAC1 complex to facilitate the formation of motile focal adhesions. These findings suggest that RGL1 can potentially serve as a therapeutic target for CRC metastasis.

Key words: Ral guanine nucleotide dissociation stimulator-like 1, metastatic colorectal cancer, motile focal adhesion, CDC42, RAC1

翁诺舟, 谭彬, 曾文涛, 古家宇, 翁炼基, 郑克鸿. 过表达RGL1通过激活CDC42/RAC1复合体上调运动型黏着斑组装促进结直肠癌转移[J]. 南方医科大学学报, 2025, 45(5): 1031-1038.

Nuozhou WENG, Bin TAN, Wentao ZENG, Jiayu GU, Lianji WENG, Kehong ZHENG. RGL1 overexpression promotes metastasis of colorectal cancer by upregulating motile focal adhesion assembly via activating the CDC42/RAC1 complex[J]. Journal of Southern Medical University, 2025, 45(5): 1031-1038.

图/表 5

图1 RGL1在转移性结直肠癌中高表达

Fig.1 RGL1 is highly expressed in metastatic colorectal cancer (CRC). A: Expression of RGL1 in non-metastatic CRC (nmCRC) and metastatic CRC (mCRC) based on data from GSE39582 and GSE87211 datasets. B: RGL1 mRNA levels in clinical specimens of nmCRC and mCRC. C: Immunohistochemical staining of RGL1 in nmCRC and mCRC (Original magnification: ×400). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图2 构建RGL1过表达细胞和敲低细胞

Fig.2 Construction of RGL1-overexpressing and knockdown cell lines. A: Expression of RGL1 in NCM460 and CRC cell lines detected by Western blotting. B, C: Verification of RGL1 overexpression and knockdown efficiency in different CRC cell lines using Western blotting.

图3 RGL1高表达促进结直肠癌的转移与侵袭

Fig.3 Overexpression of RGL1 promotes metastasis and invasion of CRC. A: Transwell migration assay of CRC cells with RGL1 overexpression or knockdown. B: Transwell invasion assay of CRC cells with RGL1 overexpression or knockdown. C: Orthotopical tumor and liver metastasis of SW480 cell xenografts with and without RGL1 knockdown implanted in the cecum of nude mice. D: Survival curve analysis of mice in shNC and shRGL1 groups. E: HE staining of liver tissues from mice in shNC and shRGL1 groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图4 RGL1高表达促进运动型黏着斑组装

Fig.4 RGL1 overexpression promotes motile focal adhesion assembly. A: Relationship between RGL1 and focal adhesion in CRC based on data from Gene Set Enrichment Analysis (GSEA). B: Fibronectin adhesion assays for detecting adherent cells between veh and RGL1+HCT116 cells. C: FN adhesion assays for detecting adherent cells between shNC and shRGL1 SW480 cells (***P<0.001, ****P<0.0001). D: Immunofluorescence assay showing cellular expression of FAK (scale bar=10 µm). E: Immunofluorescence assay showing cellular expression of paxillin (scale bar=10 µm).

图5 RGL1通过激活CDC42/RAC1复合体促进黏着斑组装

Fig.5 RGL1 facilitates focal adhesion assembly by activating the CDC42/RAC1 complex. A: Western blotting for Rho GTPase family (CDC42, RAC1 and RhoA) in RGL1+ or shRGL1 cells. B-D: Co-immunoprecipitation assay of RhoA, RhoAGTP, CDC42, CDC42GTP, RAC1 and RAC1GTP in RGL+ or shRGL1 cells.

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