Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells

Abstract

Background: 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.

Materials and methods: 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.

Results: 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.

Conclusion: 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.

Keywords: CRISPR/Cas9; CXCL12; CXCR4; CXCR7; TNBC; triple-negative breast cancer cells.

Figure 1

Design of lentivirus, transfection, screening, and PCR sequencing of single-knockout (Sg-CXCR4 and Sg-CXCR7) and co-knockout (Sg-CXCR4+7) cell clones and verification of the protein expression level by Western blotting. (A and B) The knockout target sequence of CXCR4 is located in its exon 2, while the knockout target sequence of CXCR7 is located in exon 1. (C) In MDA-MB-231 cells, after transfection with Cas9/SgRNA lentivirus, the transfection efficiency was significant. (D–F) The PCR products of monoclonal cells after the knockout of CXCR4 and CXCR7 target genes were sequenced. (G) Western blotting was used to detect the expression of CXCR4 and CXCR7 in monoclonal cells. Abbreviations: NC, negative control; ORIG, original; UNRE, unread; FRST, first; SCND, second; nt, nucleotide; MUTA, mutation.

Figure 2

Knockout of target genes CXCR4 and CXCR7 can significantly inhibit cell proliferation and cell cycle changes. Colony formation (A and B) and CCK-8 (C) assays indicated that the knockout of CXCR4 and/or CXCR7 represses TNBC cell proliferation. Moreover, the co-knockout effect was more significant than that of single knockout. The representative images and quantification of flow cytometry analysis of cells after transfection is shown. Knockdown of CXCR4 and/or CXCR7 reduced the number of cells in the G1 phase but promoted the number of cells in the S and G2/M phases (D and E). The inhibition in the co-knockout group was more significant. Symbol “#” represents the comparison with the CXCR4+7-ko group. Symbol “*” represents the comparison with the NC group (****P<0.0001; ^####P<0.0001, ^###P<0.001, ##P<0.01). The data were analyzed in triplicate, and similar results were obtained. Representative images are illustrated. Abbreviations: CCK, Cell Counting Kit; TNBC, triple-negative breast cancer; NC, negative control.

Figure 2

Knockout of target genes CXCR4 and CXCR7 can significantly inhibit cell proliferation and cell cycle changes. Colony formation (A and B) and CCK-8 (C) assays indicated that the knockout of CXCR4 and/or CXCR7 represses TNBC cell proliferation. Moreover, the co-knockout effect was more significant than that of single knockout. The representative images and quantification of flow cytometry analysis of cells after transfection is shown. Knockdown of CXCR4 and/or CXCR7 reduced the number of cells in the G1 phase but promoted the number of cells in the S and G2/M phases (D and E). The inhibition in the co-knockout group was more significant. Symbol “#” represents the comparison with the CXCR4+7-ko group. Symbol “*” represents the comparison with the NC group (****P<0.0001; ^####P<0.0001, ^###P<0.001, ##P<0.01). The data were analyzed in triplicate, and similar results were obtained. Representative images are illustrated. Abbreviations: CCK, Cell Counting Kit; TNBC, triple-negative breast cancer; NC, negative control.

Figure 3

Knockout of the target genes CXCR4 and CXCR7 can significantly inhibit the migration ability of TNBC cells. The transwell migration assay showed that single knockout and co-knockout of CXCR4 and CXCR7 significantly inhibited the migration of TNBC cells, and co-knockout was more effective. Regardless of the control group or experimental group, the addition of chemokine CXCL12 significantly increased the migration ability of TNBC cells (A) no added CXCL12; (B) added CXCL12; (C) is an analysis of the results of graphs A and B. Wound healing showed that single knockout and co-knockout of CXCR4 and CXCR7 resulted in a significant decrease in cell migration (D and E). Symbol “*” represents the comparison with the NC group. Symbol “#” represents the comparison with the CXCR4+7-ko group (****P<0.0001; ^####P<0.0001). The data were analyzed in triplicate, and similar results were obtained. Representative images are illustrated. Abbreviations: TNBC, triple-negative breast cancer; NC, negative control; w/o, without.

Figure 4

Knockout of the target genes CXCR4 and CXCR7 can significantly inhibit the invasion ability of TNBC cells. The transwell invasion assay showed that single knockout and co-knockout of CXCR4 and CXCR7 significantly inhibited the invasion of TNBC cells, and co-knockout was more effective. Regardless of the control group or experimental group, the addition of chemokine CXCL12 significantly increased the migration ability of TNBC cells (A–C). Symbol “*” represents the comparison with the NC group. Symbol “#” represents the comparison with the CXCR4+7-ko group (****P<0.0001, ***P<0.001; ^####P<0.0001, ^###P<0.001). The data were analyzed in triplicate, and similar results were obtained. Representative images are illustrated. Abbreviations: TNBC, triple-negative breast cancer; NC, negative control, w/o, without.