| Backgroud:Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies, with 655,000 deaths worldwide per year (from the WHO), which severely threaten the health of human. In the clinical, CRC is often lethal when invasion and/or metastasis occur. According to the WHO, there are ninety percent of people whose CRC is found at an early stage are alive five years after the diagnosis. However, once the CRC has spread to nearby organs or lymph nodes, the likelihood of remaining alive five years after the diagnosis is much lower. Only 39 percent of CRCs are found at that early stage. Therefore, it is important to identify the molecular pathogenesis of the matastasis and invasion of CRC.Pinl, a member of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases), can act as a molecular switch by binding a subset of proteins phosphorylated at Ser/Thr-Pro motifs and regulating their biological activity through the isomerization of peptidyl-prolyl bonds. Recent work indicates that Pinl is an important regulator of tumorigenesis and is overexpressed or underexpressed in different tumors; but little else is known about the role of this enzyme plays in other physiological functions. This study was to explore the exact mechanism the effect of Pinl to proliferation and invasion of human Colorectal cancer SW480 and SW620 cells in vitro.Objective:1. To construct RNA inference eukaryotic expression vectors targeting to Pinl gene and investigate its inhibitory effects on Pinl expression and its activity in SW480 and SW620 cells line.2. To study the effects of down-regulation of Pinl on biological behaviors of colorectal cancer SW480 and SW620 cells and detect the states of the related migration and invasive genes after transfected recombinant plasmid.3. To study the effects of down-regulation of Pinl on the bioactivity of NF-κB and the protein expressions and bioactivities of MMP-2 and MMP-9 in SW480 and SW620 cells Methods:1. The eukaryotic expression vector of RNA interfering (shRNA) for Pinl gene (pGenesil-1-PIN1) was constructed, which was confirmed by sequencing.2. After transfected with pGenesil-1-PIN1 by LipofectamineTM 2000 Reagent, detecting the protein expression levels of Pinl, MMP-2 and MMP-9 in SW480 and SW620 cells.3. The cells motility were tested by Wound healing assay and Boyden chamber assay after transfected with pGenesil-1-PIN1 in SW480 and SW620 cells.4. The bioactivity of MMP-2 and MMP-9 were tested by Gelatin Zymography in SW480 and SW620 cells after transfected with pGenesil-1-PIN1 (SW480/p-shRNA, SW620/p-shRNA).5. The DNA-binding activity of NF-κB in the cells transfected with pGenesil-1-PIN1 were analyzed by 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 and SW620 cells transfected with pGenesil-1-PIN1, oligonucleotides containing a putative NF-κB binding site were synthesized, and EMS As were performed.Results:1. The expression plasmid against Pin1 was successfully constructed.2. Recombinant vectors could reduce the expressions of Pinl protein. In SW480 the relative protein expression of Pinl were (0.49±0.07) for SW480/p-shRNA, and (0.94±0.09) for SW480/p-CON, and (1.01±0.11) for SW 480 cells. In SW620 cells, it were 0.44±0.11 (SW620/p-shRNA),0.98±0.10(SW620/p-CON), and 0.99±0.09(SW620)relatively(P<0.05).3. Recombinant vectors could reduce the expressions of MMP-2 and MMP-9 protein. SW480/p-shRNA were (0.39±0.08) and (0.45±0.07), SW480/p-CON were (0.82±0.07) and (0.83±0.07), SW480 were (0.84±0.07) and (0.93±0.05). SW620/p-shRNA were (0.32±0.04) and (0.41±0.09), SW620/p-CON were (0.76±0.03) and (0.94±0.07), SW620 were (0.76±0.01) and (0.95±0.05) (P<0.05).4. After transfected with pGenesil-1-PIN1, the metastasis ability was highly weakened. The cell migration and cell invasion ability was detected by Wound healing assay and Boyden chamber assay. Boyden Chamber assay results displayed that the cells motility 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). And (96.4±3.9) per field (×10 objective) to (52.7±4.4) per field (P<0.05, Student's t-test) for SW620 cells transfected with pGenesil-1-PIN1 (SW620/p-shRNA).5. EMS A results revealed that low DNA-binding activity of NF-κB in p-shRNA cells compared to p-CON cells, and showed that NF-κB bind directly to the oligonucleotides which containing putative NF-κB binding sites in the MMP-9 promoter derived from the genomic DNA of p-shRNA cells.Conclusion:1. RNA interference expression vector of targeting to Pinlis successfully constructed and it can inhibite the expression and activity of Pinl, it prepared for exploring the function of Pinl gene in CRC with RNA interference(RNAi) technique.2. Silencing the Pinl expression by RNAi could inhibit the migration and cell invasion ability of SW480 and SW620 cells, which was probably related with Down-regulating the expression of MMP-2 and MMP-9 due to the decrease NF-κB transcriptional activity. It may provide a novel therapeutic approach for color cancer therapy.
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