The influence of lentivirus-mediated CXCR4 RNA interference on hepatic metastasis of colorectal cancer

Abstract

The aim of this study was to construct a lentiviral vector of CXCR4-siRNA (Lenti-CXCR4-siRNA) and investigate whether the vector can inhibit the growth, migration, invasion and hepatic metastasis of colorectal cancer (CRC). RT-PCR and western blotting were employed to identify the ideal RNA interference sequence. Lenti-CXCR4-siRNA was constructed and transfected into the SW480 cell line. We used RT-PCR and western blotting to measure the expression of CXCR4 RNA and protein, respectively; the MTS assay to assess the proliferation of SW480 cells; transwell chambers to estimate the inhibitory effect on migration and invasion; and the Balb/c nude mouse model of CRC to examine the inhibition of hepatic metastasis. The relative expression of the CXCR4 gene and protein was 5.4 and 18.95%, respectively, in the siCXCR4 group. The genes in the expression plasmid pLenti-CXCR4-siRNA were in the correct order. In the SW480, nonsense control (NC) and the Lenti-CXCR4-siRNA groups CXCR4 RNA levels were, respectively, 0.54±0.06, 1.00±0.03 and 0.11±0.04 (P=0.0001); CXCR4 protein levels were 0.60±0.03, 0.72±0.03 and 0.18±0.02 (P=0.0001); the OD value was 1.38±0.04 (P=0.0050), 1.28±0.05 (P=0.0256) and 0.92±0.06; SW480 cell number in migration test was 32±6.85, 32.63±1.69 and 0.75±0.71 (P=0.0000); SW480 cell number in the invasion test was 29.13±10.3, 30.38±6.09 and 0.63±0.74 (P=0.0000); hepatic metastasis number was 7.10±3.98 (P=0.034), 7.50±4.09 (P=0.019) and (3.50±2.51); hepatic metastasis mean weight (in g) was 2.25±2.51 (P=0.000), 2.11±2.38 (P=0.000) and 1.45±2.07. Lenti-CXCR4-siRNA constructs were correctly constructed and effectively inhibit the expression of CXCR4 RNA and protein, reducing the proliferation, migration, invasion capacity of SW480 cells and hepatic metastasis of CRC.

Figure 1.

Cells in the siCXCR4 group had the lowest CXCR4 expression.

Figure 2.

Identification by cutting: M, 1 kb DNA ladder; 1, blank GV115; 2, pLenti-CXCR4-siRNA was cut by XhoI restriction enzyme into the linear form.

Figure 3.

The sequence of pLenti-CXCR4-siRNA was identical to the designed sequence.

Figure 4.

pLenti-CXCR4-siRNA transfected cells show green fluorescence (×100).

Figure 5.

Lenti-CXCR4-siRNA infected SW480 cells show green fluorescence (×100).

Figure 6.

Protein expression of CXCR4: 1, the SW480 group; 2, NC group; 3, Lenti- CXCR4-siRNA group. Cells in the Lenti-CXCR4-siRNA group had the lowest CXCR4 protein expression.

Figure 7.

Migration experiment: (A) SW480 group; (B) NC group; (C) Lenti-CXCR4-siRNA group (×100). The number of migrated cells was significantly lower in the Lenti-CXCR4-siRNA group than in the SW480 group and NC group.

Figure 7.

Migration experiment: (A) SW480 group; (B) NC group; (C) Lenti-CXCR4-siRNA group (×100). The number of migrated cells was significantly lower in the Lenti-CXCR4-siRNA group than in the SW480 group and NC group.

Figure 7.

Migration experiment: (A) SW480 group; (B) NC group; (C) Lenti-CXCR4-siRNA group (×100). The number of migrated cells was significantly lower in the Lenti-CXCR4-siRNA group than in the SW480 group and NC group.