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

Abstract

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 confi rmed 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 blott ing 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 fi eld (× 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 blott ing 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 eff ect on the expression and bioactivity of MMP-9 in colon cancer SW480 cells, possibly through the transcription factor NF-κB.

Knockdown of Pin1 leads to reduced angiogenic potential and tumorigenicity in glioblastoma cells

“…Previous studies have established that MMP9 expression is associated with glioblastoma growth and invasion (30). Recently, it has been demonstrated that the inhibition of Pin1 in colorectal carcinoma cells decreases the level and enzymatic activity of MMP9, presumably via nuclear factor-κB signaling (13). Similarly, a reduction in MMP9 following Pin1 inhibition was observed in the present study.…”

Induction of matrix metalloproteinase-2 and -9 via Erk1/2-NF-κB pathway in human astroglia infected with Toxoplasma gondii

“…Nuclear extracts (10 g) were incubated with 300 ng biotin 3 -end labeled double-strand oligonucleotide probe (GeneMark Technology Co., Ltd., Taichung, Taiwan), 2 g of poly deoxyinosinic-deoxycytidy acid (dI-dC), 1 L 50% glycerol, and 4 L 5× DNA loading dye for 30 min at room temperature. The following biotin 3 -end labeled double-strand oligonucleotide probes were used in this study: commercial consensus oligonucleotide sequence of MMP-9-NF-B (5 -TGC CCC AGT GGA ATT CCC CAG CCT TG-3 ) (Farina et al, 1999;Qin et al, 2010). Protein-DNA complexes were detected by running the samples on non-denaturing 6% (w/v) PAGE in 0.5× Tris-borate-EDTA (TBE) buffer (GeneMark Technology Co., Ltd.), transferred to positively charged nylon membranes (Millipore Corporation, Billerica, MA, USA) using a blot transfer apparatus, and then cross-linked using a Stratagene cross-linker.…”

Anti-Diabetic Atherosclerosis by Inhibiting High Glucose-Induced Vascular Smooth Muscle Cell Proliferation via Pin1/BRD4 Pathway

“…When VSMCs switch from the contractile phenotype to the proliferative migration phenotype, MMPs are secreted and inflammatory cytokines are produced to promote vascular remodeling [ 31 ]. Studies have shown that Pin1 and RBD4 interact with NF- κ B and cause increased expression of MMP genes [ 32 , 33 ]. Our study showed that juglone and JQ1 inhibited increased MMP-9 protein expression induced by high glucose, which is consistent with studies by Liang and Duan et al [ 34 , 35 ].…”