CXCL12 gene silencing down-regulates metastatic potential via blockage of MAPK/PI3K/AP-1 signaling pathway in colon cancer

Abstract

Background: To investigate the effect of CXCL12 gene silencing on proliferation,invasion, angiogenesis and the relationship of MAPK/PI3K/AP-1 signaling pathway in colon cancer cells.

Methods: RT-PCR and Western-blot were used to detect the expression of CXCL12 mRNA and protein in four colon cancer cell lines. Human colon cancer cells were transfected with CXCL12 siRNA carrying by Lipofectamine 2000. The expression of CXCL12 protein was confirmed by immunoblotting. WST-1, invasion and angiogenesis assay were used to examine the effect on proliferation, invasion and angiogenesis in colon cancer cells after CXCL12 siRNA silence, respectively. The phosphorylation of MAPK/PI3K/AP-1 protein levels was detected by Western blotting in CXCL12 siRNA suppression DLD-1 cell.

Results: CXCL12 mRNA and proteins were only expressed in DLD-1 colon cancer cell lines. CXCL12 siRNA were transfected into DLD-1 cells, the expression CXCL12 proteins was significantly inhibited (P < 0.01), and the proliferation, invasion and angiogenesis of DLD-1 cells were inhibited significantly (P < 0.01). CXCL12 gene silencing resulted in blockage of MAPK, PI3K and AP-1 phosphorylation by CXCL12-induced in DLD-1 colon cancer cell.

Conclusion: The silencing CXCL12 gene significantly inhibits the proliferation, invasion and angiogenesis ability of some types colon carcinoma cells through down-regulation of MAPK/PI3K/AP-1 signaling pathway.

Keywords: CXCL12 siRNA; Colon cancer; Invasion; MAPK/PI3K/AP-1 signaling pathway; Proliferation.

Fig. 1

Expression of CXCL12 and CXCR4 in colon cancer cell lines. a Detection of CXCL12 and CXCR4 mRNA in colon cancer cells. PCR products stained with ethidium bromide were subjected to 1.5% agarose gel electrophoresis. b The protein expression of CXCL12 and CXCR4 in colon cancer cell lines was confirmed by Western blotting analysis. β-Actin served on a loading control

Fig. 2

The expression of CXCL12 protein in colon cancer cell line after silencing of CXCL12 gene. Knockdown of CXCL12 by CXCL12 siRNA was confirmed by immunoblotting (a) in all four colon cancer cell lines. siRNA duplex oligoribonucleotides were transfected into cells for 48 h; the total RNA and proteins were extracted and then western blot. The grayscale values of the strips were measured by Image J software (b). Multiple comparisons were performed by one-way ANOVA followed by SNK test. Values are expressed as mean ± SD. Bars indicated SD

Fig. 3

The effect of CXCL12 gene silencing on the proliferation of colon cancer cells. a DLD-1, HT-29 CaCo-2 and Colo320 cells were transfected by CXCL12 siRNA and Control siRNA for 24 h; the proliferation of cancer cells was measured by WST-1 assay. The proliferation of DLD-1 cells in CXCL12 siRNA group was significantly inhibited (compared with the untransfected and control siRNA groups, P < 0.01, respectively). b The cell proliferation curve showed that there was no significant difference in the proliferation of DLD-1 colon cancer cells before 24 h; the proliferation of CXCL12 siRNA group was significantly lower than those in the untransfected and control siRNA groups after 48, 72, 96, 120 h (P < 0.01). Multiple comparisons used the method of one-way ANOVA and followed by the SNK test. Values are expressed as mean ± SD. Bars indicated SD, *P < 0.01

Fig. 4

The effect of CXCL12 gene silencing on the invasive ability of colon cancer cells. A DLD-1 and HT-29 invasion by influence of CXCL12 siRNA was assessed by the BD Bio-Coat Matrigel invasion assay. The cells were incubated for 24 h; the invading cells were fixed and stained with Diff-Quick stain. The invading cells were counted in five random microscopic fields (×200). The invasive ability of DLD-1 transfected with CXCL12 siRNA group was significantly decreased compared with the untransfected and control siRNA groups (P < 0.01). The invasive ability of HT-29 that transfected CXCL12 siRNA group had no significant change compared with the untransfected and control siRNA groups. B The invasive figure is as follows: a DLD-1 cells untreated; b DLD-1 cells Control siRNA; c DLD-1 cells CXCL12 siRNA; d HT-29 cells untreated; e HT-29 cells Control siRNA; f HT-29 cells CXCL2 siRNA. Multiple comparisons used the method of one-way ANOVA and followed by the SNK test. Columns, relative invading number. Bars indicate SD, *P < 0.01

Fig. 5

CXCL12 gene silencing influence on tube formation by HUVEC. Colon cancer cells were co-cultured with HUVEC and fibroblast using double-chamber method. All cells were cultured for total 11 days. The tubular formation was stained with anti-CD31 antibody by the protocols of manufacturer. The area of tubular formation was measured quantitatively over ten different fields for each condition using an image analyzer. A The image of angiogenesis shows a DLD-1 cells untreated; b DLD-1 cells Control siRNA; c DLD-1 cells CXCL2 siRNA; d HT-29 cells untreated; e HT-29 cells Control siRNA; f HT-29 cells CXCL12 siRNA. B The tubular formation of HUVEC was significantly inhibited by co-culture with CXCL12 siRNA DLD-1 cells compared with untransfected and control siRNA groups, respectively (P < 0.01). HT-29 of CXCL12 siRNA group had no significant change compared with the untransfected and control siRNA groups. Multiple comparisons used the method of one-way ANOVA and followed by the SNK test. Bars indicate SD, P < 0.01

Fig. 6

Effects of CXCR4 siRNA on phosphorylation of major proteins in MAPK/PI3K/AP-1 signaling pathway. Colon cancer cells were stimulated by different concentrations of CXCL12 for 15 min. The proteins were extracted and separated by SDS-PAGE, transferred to membranes, and the membranes probed with antibody directed against phospho-MAPK, phospho-PI3K, phospho-AP-1, total MAPK, total PI3K and total AP-1. After different concentrations of CXCL12 were used to stimulate the DLD-1 CXCR4 siRNA, untransfected, control siRNA groups and HT-29 cells for 15 min, the effects of CXCL12 on phosphorylation of member proteins in MAPK/PI3K/AP-1 signaling pathway were detected by Western blot. a The phosphorylation levels of MAPK, PI3K and AP-1 proteins in CXCR4 siRNA group after being simulated by different concentrations of CXCL12 were significantly weaker than the untransfected and control siRNA groups. b The phosphorylation of MAPK, PI3K and AP-1 proteins were positively correlated with the concentration of CXCL12 in HT-29 cells