Silencing Pin1 suppresses the expression and bioactivity of MMP-9 through NF-κB in colorectal carcinoma SW480 cells

Objective To examine the effect of Pin1 on the expression and bioactivity of MMP-9 through NF-κB in human colorectal carcinoma SW480 cells. Methods The eukaryotic expression vector of RNA interfering (shRNA) with the Pin1 gene (pGenesil-1-PIN1) was constructed in our previous experiments and was confirmed through sequencing. Cell motility was tested through the wound healing assay and the Boyden chamber assay. The protein levels and bioactivity of MMP-9 were tested by Western blotting and gelatin zymography in SW480 cells after transfection with pGenesil-1-PIN1 (SW480/p-shRNA). The DNA-binding activity of NF-κB in cells transfected with pGenesil-1-PIN1 was analyzed by the electrophoretic mobility shift assay (EMSA). In addition, to determine whether NF-κB has direct interaction with the MMP-9 promoter derived from the genomic DNA of SW480 cells transfected with pGenesil-1-PIN1, oligonucleotides containing a putative NF-κB binding site were synthesized and EMSAs were performed. Results The results of the Boyden chamber assay showed that cell motility was reduced from 90.2 ± 6.5 per field (× 10 objective) to 49.6 ± 7.2 per field ( P < 0.05, Student’s t -test) for SW480 cells transfected with pGenesil-1-PIN1 (SW480/p-shRNA). Western blotting detected low protein levels of Pin1 and MMP-9 in SW480/p-shRNA cells. The relative protein levels of Pin1 were 0.49 ± 0.07 in SW480/p-shRNA compared with 0.94 ± 0.09 in SW480/p-Con, and MMP-9 were 0.45 ± 0.07 in SW480/p-shRNA, 0.83 ± 0.07 in SW480/p-Con ( P < 0.05). The results of gelatin zymography showed that silencing Pin1 markedly reduced the bioactivity of MMP-9 in SW480 cells. EMSA results revealed low DNA-binding activity of NF-κB in SW480/p-shRNA cells compared to SW480/p-Con cells, and that NF-κB bound directly to the oligonucleotides which contained putative NF-κB binding sites in the MMP-9 promoter derived from the genomic DNA of SW480/p-shRNA cells. Conclusion Inhibited Pin1 expression may contribute to the suppressive effect on the expression and bioactivity of MMP-9 in colon cancer SW480 cells, possibly through the transcription factor NF-κB.