CXCR4/CXCR7与NF-κB通路在三阴性乳腺癌中的关联及功能研究

Association and functional role of CXCR4/CXCR7 and the NF-κB pathway in triple-negative breast cancer

摘要:

背景与目的 三阴性乳腺癌（TNBC）具有高度侵袭性，且缺乏有效的靶向治疗手段。趋化因子受体CXCR4和CXCR7在TNBC中表达升高，可能通过激活NF-κB信号通路促进肿瘤细胞的迁移与侵袭。本研究旨在探讨CXCR4/CXCR7及NF-κB信号通路在TNBC细胞迁移和侵袭中的作用机制。方法 在TNBC MDA-MB-231细胞中，采用CRISPR/Cas9技术分别或同时敲除CXCR4和CXCR7基因，并设立NF-κB抑制剂BAY 11-7082处理组。通过Western blot检测IκB-α和p65的磷酸化水平评估NF-κB通路活性；使用CCK-8实验、划痕实验和Transwell实验评估各组细胞的增殖、迁移及侵袭能力。结果 在成功构建CXCR4、CXCR7单基因敲除及双基因敲除的MDA-MB-231细胞株后，Western blot结果显示，这些敲除明显降低了NF-κB信号通路关键蛋白IκB-α与p65的磷酸化水平（均P<0.05），其中CXCR4/CXCR7双敲除组抑制效果较单敲组更明显，但NF-κB通路抑制剂BAY 11-7082（5 μmol/L，24 h）对IκB-α与p65磷酸化的抑制程度亦优于双基因敲除组（均P<0.05）。划痕实验和Transwell迁移/侵袭实验结果表明，无论是基因敲除还是NF-κB通路抑制均可降低TNBC细胞的迁移和侵袭能力（均P<0.05）。其中，CXCR4/CXCR7双敲除组的迁移与侵袭抑制程度明显优于单基因敲除组，而BAY 11-7082处理组表现出最强的抑制效果，迁移率与侵袭细胞数均明显低于CXCR4/CXCR7双敲组（均P<0.05）。结论 CXCR4/CXCR7通过激活NF-κB信号通路促进TNBC细胞的迁移与侵袭，提示NF-κB信号通路可能是TNBC联合免疫治疗的潜在靶点。

Abstract:

Background and Aims Triple-negative breast cancer (TNBC) is highly aggressive and lacks effective targeted therapies. The chemokine receptors CXCR4 and CXCR7 are overexpressed in TNBC and may promote tumour cell migration and invasion by activating the NF-κB signalling pathway. This study aimed to investigate the roles of CXCR4/CXCR7 and the NF-κB pathway in regulating the migration and invasion of TNBC cells.Methods In the TNBC cell line MDA-MB-231, CRISPR/Cas9 technology was used to individually or in combination knock out the CXCR4 and CXCR7 genes. Additionally, a group treated with the NF-κB inhibitor BAY 11-7082 was established. The phosphorylation levels of IκB-α and p65 were assessed by Western blotting to evaluate NF-κB pathway activity. Cell proliferation, migration, and invasion were evaluated using the CCK-8 assay, wound healing assay, and Transwell assay, respectively.Results MDA-MB-231 cell lines with CXCR4, CXCR7, or dual gene knockout were successfully established. Western blot analysis revealed that the phosphorylation levels of IκB-α and p65 were significantly reduced in all knockout groups (all P<0.05), with the dual knockout group exhibiting a more substantial inhibitory effect than the single knockouts. However, BAY 11-7082 (5 μmol/L, 24 h) exerted a more pronounced suppression of IκB-α and p65 phosphorylation compared to the dual knockout group (all P<0.05). Functional assays demonstrated that both gene knockout and NF-κB inhibition significantly impaired the migration and invasion of TNBC cells (all P<0.05). Among all groups, the dual knockout of CXCR4 and CXCR7 showed greater inhibitory effects than either single knockout. At the same time, the BAY 11-7082 treatment exhibited the most potent suppression of both migration and invasion (both P<0.05).Conclusion CXCR4 and CXCR7 promote TNBC cell migration and invasion by activating the NF-κB signalling pathway, suggesting that the NF-κB pathway may serve as a potential therapeutic target for combination immunotherapy in TNBC.