RAB1A在乳腺癌中的表达特点及其对乳腺癌转移的促进作用

黄愉棉; 李世昂; 李宇翔; 孙刚

doi:10.3971/j.issn.1000-8578.20260157

RAB1A在乳腺癌中的表达特点及其对乳腺癌转移的促进作用

The expression character of RAB1A in breast cancer and its promotion of breast cancer metastasis

摘要

摘要: 目的探究RAB1A在乳腺肿瘤微环境（Tumor microenvironment, TME）中的表达特征及其通过调控成纤维细胞功能促进肿瘤转移的作用机制，寻找靶向TME的抗转移治疗潜在靶点。方法对16例三阴性乳腺癌（Triple-negative breast cancer, TNBC）及HER2过表达乳腺癌患者的肿瘤与癌旁组织进行蛋白组测序。对4例TNBC及HER2过表达乳腺癌患者穿刺样本进行单细胞转录组测序（Single-cell RNA sequencing, scRNA-seq）。从蛋白组层面及单细胞转录组层面分析TME细胞异质性及RAB1A表达模式。使用ELISA及划痕实验等验证RAB1A调控肿瘤相关成纤维细胞（cancer associated fibroblasts, CAFs）功能，进而促进三阴性乳腺癌细胞转移的机制。结果 RAB1A特异性高表达于乳腺癌患者的CAFs细胞群体，功能模块上显示与促转移的“分泌途径”和“基质重塑”模块相关。将人乳腺成纤维细胞（Human Mammary Fibroblasts，HMF）、CAFs、CAF-OE-RAB1A与MDA-MB-231细胞共培养24h后，划痕实验显示24小时细胞迁移率分别为：MDA-MB-231 NC组25.07%、MDA-MB-231&HMF组30.56%、MDA-MB-231&CAFs组38.86%、MDA-MB-231&CAF-OE-RAB1A组47.21%。 CAFs共培养组比NC组（p <0.01)、HMF共培养组（p <0.05)的MDA-MB-231细胞迁移率显著提升。OE-RAB1A共培养组相比CAFs共培养组则进一步提升（p<0.05）。收集24h共培养上清进行TGF-b1 ELISA检测：HMF组29.52pg/ml、CAFs组417.62pg/ml、CAF-OE-RAB1A组1030.56pg/ml。OE-RAB1A组相比于CAFs组（p<0.001)和HMF组分泌TGF-b1（p<0.0001)显著增多。CAFs组相比于HMF组分泌TGF-b1显著增多（p<0.01）。结论 RAB1A特异性高表达于乳腺癌TME中的CAFs细胞群，可调控CAFs分泌TGF-b1增多进而促进乳腺癌转移。这一特性使其可能成为靶向TME抗转移治疗的有效靶点。

Abstract: Objective To investigate the expression characteristics of RAB1A in the breast tumor microenvironment (TME) and its mechanism of promoting tumor metastasis by regulating fibroblast function, with the aim of identifying potential targets for TME-targeted anti-metastatic therapy. Methods Proteomic sequencing was performed on tumor and adjacent tissues from 16 patients with triple-negative breast cancer (TNBC) or HER2-overexpressing breast cancer. Single-cell RNA sequencing (scRNA-seq) was conducted on biopsy samples from 4 patients with TNBC or HER2-overexpressing breast cancer. TME cell heterogeneity and RAB1A expression patterns were analyzed at both the proteomic and single-cell transcriptomic levels. ELISA and wound healing assays were utilized to verify the mechanism by which RAB1A regulates the function of cancer-associated fibroblasts (CAFs), thereby promoting the metastasis of TNBC cells. Results RAB1A was specifically highly expressed in the CAFs. Functional module analysis indicated an association with pro-metastatic “secretion pathway” and “matrix remodeling” modules. After co-culturing Human Mammary Fibroblasts (HMF), CAFs, and the CAFs of RAB1A-overexpressing (CAF-OE-RAB1A) with MDA-MB-231 cells for 24 hours, the wound healing assay showed the following 24-hour cell migration rates: 25.07% in the MDA-MB-231 NC group, 30.56% in the MDA-MB-231 & HMF group, 38.86% in the MDA-MB-231 & CAFs group, and 47.21% in the MDA-MB-231 & CAF-OE-RAB1A group. The migration rate of MDA-MB-231 in the CAFs co-culture group was significantly higher than that in the NC group (p<0.01) and the HMF co-culture group (p<0.05). The migration rate was further significantly increased in the OE-RAB1A co-culture group (p<0.05). Supernatants from the 24-hour co-cultures were collected for TGF-b1 ELISA detection, yielding the following results: 29.52 pg/ml in the HMF group, 417.62 pg/ml in the CAFs group, and 1030.56 pg/ml in the CAF-OE-RAB1A group. TGF-b1 secretion in the OE-RAB1A group was significantly higher than that in the CAFs group (p<0.001) and the HMF group (p<0.0001). TGF-b1 secretion in the CAFs group was significantly higher than that in the HMF group (p<0.01). Conclusion RAB1A is specifically expressed in CAFs of breast cancer and regulates CAFs to secrete more TGF-b1, thereby promoting breast cancer metastasis. This characteristic makes it a potential effective target for TME-targeted anti-metastatic therapy.

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