Interfering with CXCR4 expression inhibits proliferation, adhesion and migration of breast cancer MDA-MB-231 cells

Abstract

To investigate the effect and mechanism of the CXC chemokine receptor 4 (CXCR4) in the proliferation and migration of breast cancer, a short-hairpin RNA (shRNA) eukaryotic expression vector targeting CXCR4 was constructed, and the impact of such on the proliferation, adhesion and migration of human breast cancer MDA-MB-231 cells was observed. The fragments of CXCR4-shRNA were synthesized and cloned into a pGCsi-U6-Neo-green fluorescent protein vector. The recombinant plasmids were transfected into 293T cells and the most efficacious interfering vector was selected. MDA-MB-231 cells were transfected by liposome assay. The effects of silencing CXCR4 expression by shRNA on the growth, adhesion and migration of MDA-MB-231 cells were determined by Cell Counting Kit-8, cell-matrix adhesion and wound-healing assays. The shRNA eukaryotic expression vectors targeting CXCR4 (CXCR4-shRNA) were successfully constructed and transfected into 293T cells. Quantitative polymerase chain reaction and western blot analysis revealed that the maximum inhibitory rate of CXCR4 expression was 81.3%. CXCR4-shRNA transfection significantly inhibited the proliferation of MDA-MB-231 cells (P<0.05), as well as the adhesion between MDA-MB-231 cells and the extracellular matrix (P<0.05). Furthermore, wound-healing assays demonstrated that the migration distance of MDA-MB-231 cells in the CXCR4-shRNA transfection group was significantly smaller than that in the control plasmid and blank control groups (P<0.01). The CXCR4-shRNA interfering vector specifically inhibited CXCR4 expression, as well as the proliferation, adhesion and migration of MDA-MB-231 cells.

Keywords: CXC chemokine receptor 4; RNA interference; breast cancer.

Figure 1

Identification of recombinant CXCR4 interfering RNA plasmid by restriction enzyme digestion. M, marker; 1, pGCsi-CXCR4-1/ShRNA; 2, pGCsi-CXCR4-2/ShRNA; and 3, negative control. CXCR4, CXC chemokine receptor 4.

Figure 2

Transfection efficiency of interfering RNA CXCR4 vector in 293T cells (magnification, ×100). (A) pGCsi-CXCR4-1/ShRNA, (B) pGCsi-CXCR4-2/ShRNA and (C) negative control. CXCR4, CXC chemokine receptor 4.

Figure 3

(A) mRNA and (B) protein expression of CXCR-4 in 239T cells by RNA interference. 1, pGCsi-CXCR4-1/ShRNA; 2, pGCsi-CXCR4-2/ShRNA; 3, negative control; and 4, blank control. CXCR4, CXC chemokine receptor 4.

Figure 4

Silencing CXCR4 expression inhibits the proliferation of MDA-MB-231 cells. ^*P<0.05, vs. negative control or blank control. 1, pGCsi-CXCR4-1/ShRNA; 2, negative control; and 3, blank control. CXCR4, CXC chemokine receptor 4.