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. 2014 Jun;44(6):1861-9.
doi: 10.3892/ijo.2014.2348. Epub 2014 Mar 19.

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

Tian-Bao Wang  1 , Bao-Guang Hu  2 , Da-Wei Liu  3 , Han-Ping Shi  1 , Wen-Guang Dong  1
Affiliations

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

Tian-Bao Wang et al. Int J Oncol. 2014 Jun.

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.

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Figures

Figure 1.
Figure 1.
Cells in the siCXCR4 group had the lowest CXCR4 expression.
Figure 2.
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.
Figure 3.
The sequence of pLenti-CXCR4-siRNA was identical to the designed sequence.
Figure 4.
Figure 4.
pLenti-CXCR4-siRNA transfected cells show green fluorescence (×100).
Figure 5.
Figure 5.
Lenti-CXCR4-siRNA infected SW480 cells show green fluorescence (×100).
Figure 6.
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.
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.
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.
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.
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