Yang Meng, Zeng Chen, Li Peiting, Qian Liyuan, Ding Boni, Huang Lihua, Li Gang, Jiang Han, Gong Ni, Wu Wei
Department of Breast Thyroid Surgery, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, People's Republic of China,
Central Laboratory, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, People's Republic of China.
Onco Targets Ther. 2019 May 17;12:3849-3858. doi: 10.2147/OTT.S195661. eCollection 2019.
Breast cancer is one of the most common malignancies threatening women's health. Triple-negative breast cancer (TNBC) is a special type of breast cancer with high invasion and metastasis. CXCL12 and its receptors CXCR4 and CXCR7 play a crucial role in the progress of breast cancer. The aim of this study was to investigate the effect of CXCR4 and CXCR7 on the function of TNBC.
We used the CRISPR/Cas9 technique to carry out a single knockout of the CXCR4 or CXCR7 gene and co-knockout of CXCR4 and CXCR7 genes in the TNBC cell line (MDA-MB-231). The single knockout and co-knockout cells were screened and verified by PCR sequencing and Western blot assay, the effect of single knockout and co-knockout on the proliferation of TNBC cells was examined using the Cell Counting Kit-8 and colony formation assays, the migration and invasion of TNBC cells were examined by the transwell and wound-healing assays, the changes in the cell cycle distribution after knockout were detected by flow cytometry, and the difference in the migration and invasion of single knockout and co-knockout induced by CXCL12 was observed by adding CXCL12 in the experimental group.
The single knockout of the CXCR4 or CXCR7 gene significantly reduced the cell proliferation, growth, migration, and invasion and delayed the conversion of the G1/S cycle, while the co-knockout inhibited these biological abilities more significantly. In both the knockout and control groups, the migration and invasion of CXCL12-added cells were significantly stronger than those of the non-CXCL12-added cells, and CXCL12 induced lesser migration and invasion in the CXCR4 and CXCR7 co-knockout group than in the single knockout groups.
The knockout of the CXCR4 and CXCR7 genes affects the binding capacity and functions of CXCL12, inhibits the malignant progression of TNBC cells significantly, and may become a potential target for the treatment of TNBC.
乳腺癌是威胁女性健康的最常见恶性肿瘤之一。三阴性乳腺癌（TNBC）是一种具有高侵袭和转移能力的特殊类型乳腺癌。CXCL12及其受体CXCR4和CXCR7在乳腺癌进展中起关键作用。本研究旨在探讨CXCR4和CXCR7对TNBC功能的影响。
我们使用CRISPR/Cas9技术在TNBC细胞系（MDA-MB-231）中对CXCR4或CXCR7基因进行单敲除以及对CXCR4和CXCR7基因进行共敲除。通过PCR测序和蛋白质印迹分析对单敲除和共敲除细胞进行筛选和验证，使用细胞计数试剂盒-8和集落形成试验检测单敲除和共敲除对TNBC细胞增殖的影响，通过Transwell和伤口愈合试验检测TNBC细胞的迁移和侵袭能力，通过流式细胞术检测敲除后细胞周期分布的变化，并在实验组中添加CXCL12观察单敲除和共敲除诱导的迁移和侵袭差异。
CXCR4或CXCR7基因的单敲除显著降低了细胞增殖、生长、迁移和侵袭能力，并延迟了G1/S周期转换，而共敲除对这些生物学能力的抑制作用更显著。在敲除组和对照组中，添加CXCL12的细胞的迁移和侵袭能力均明显强于未添加CXCL12的细胞，并且CXCL12在CXCR4和CXCR7共敲除组中诱导的迁移和侵袭比在单敲除组中更小。
CXCR4和CXCR7基因的敲除影响CXCL12的结合能力和功能，显著抑制TNBC细胞的恶性进展，可能成为TNBC治疗的潜在靶点。
