Moreover, the results of xenograft experiments indicated that the PAR2 inhibitor can enhance cell killing by 5-FU in vivo and suppress EMT signaling. vivo and suppress EMT signaling. Our results reveal that the TGF- effects require the coordinating action of PAR2, suggesting that PAR2 inhibition could be a new therapeutic strategy to combat 5-FU resistance in CRC. siRNA or control scrambled siRNA (Invitrogen, Carlsbad, CA, USA). To generate an expression plasmid encoding full-length PAR2, full-length human PAR2 cDNA was amplified by PCR using the primers 5-caccatgcggagccccagcgcggcgt-3 (forward) and 5-ataggaggtcttaacagtggttgaa-3 (reverse), and cloned into pcDNA3.1 Directional TOPO expression plasmid (Invitrogen). The transfection of siRNAs or plasmids was conducted using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. RNA Isolation and Quantitative-PCR (qPCR) Assay Total RNA was extracted using Tri-Reagent (Sigma-Aldrich, St. Louis, MO, USA) and reverse transcripted (Invitrogen), and qPCR was conducted using a CFX96 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The value measured for each gene was normalized relative to that of -actin gene. The sequences of the primers used in qPCR have been reported previously12. Cell Migration Analysis Cell migration assays were performed in a 24-well Transwell system featuring 8-m pore size membrane inserts (Corning, Corning, NY, USA). Briefly, 2??104 cells were transfected, starved, and trypsinized before plating in serum-free medium in the upper chamber. The lower chamber was filled with the same medium including 10% FBS. The cells in the upper chamber were wiped off using cotton swabs, and the cell that had crossed to the opposite Angiotensin III (human, mouse) side of the membrane, the invaded or migrated cells were fixed with methanol, stained with 5% crystal violet, and quantified under a microscope (Olympus Co., Shinjuku, Tokyo, Japan). Western Blotting Analysis Total cell lysates were processed in radioimmunoprecipitation assay buffer that included Boehringers protease inhibitor mixture and phosphatase inhibitors (e.g., NaF, PMSF, and sodium orthovanadate). After determining protein concentrations using Bio-Rads Bradford assay, proteins were C1qdc2 separated using SDS-PAGE, transferred to polyvinylidene fluoride membranes, and Western blotted13. Mouse Xenograft Model and Treatment All animal experiments in this study adhered to globally accepted guiding principles of animal use and care in laboratories. Female nude mice (aged 5C6 weeks old; Vital River, P.R. China) were raised under sterile conditions, and 1??106 HCT116 cells (suspended in 100 l of phosphate-buffered saline) were administered subcutaneously into each side of the mice. As soon as tumors were detected, the mice were intraperitoneally injected with 5-FU (25 mg/kg), ENMD-547 (2 mg/kg), or their combination once every 2 days for 10 consecutive days. Tumor dimensions were measured using calipers, and cancer development was quantified using the equation: 0.5??length??width2. The Angiotensin III (human, mouse) dissected tumors were fixed with and immunostained for activated caspase-3, E-cadherin, and vimentin. All procedures and experiments involving animals in this study were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the First Affiliated Hospital of Soochow University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Statistical Analysis Students t-test was used for comparisons of two independent groups; differences were considered significant at a value of p?0.05. RESULTS TGF- Signaling and PAR2 Expression Were Induced After 5-FU Treatment in CRC Patients TGF- and PAR2 levels are reported to be highly correlated with CRC development14,15; thus, we first performed qRT-PCR to examine TGF- and PAR2 mRNA expression in 46 pairs of randomly Angiotensin III (human, mouse) selected tumor tissues before and after neoadjuvant chemotherapy. After 5-FU chemotherapy, TGF- and PAR2 expression levels were significantly higher than before therapy (p?0.001) (Fig. 1A). Because TGF- and PAR2 expression is also related to cancer metastasis, we further measured their expression in metastatic cancers. We defined CRC tissues showing metastasis, tumor invasion into bile duct, and venous infiltration as aggressive CRC tissues, and found that TGF- and PAR2 were markedly upregulated in aggressive CRC tissues compared with the levels in nonaggressive tissues (p?0.01) (Fig. 1B). We also examined the expression of genes related to migration and invasion, such as genes encoding matrix metalloproteinase-2 (MMP-2), MMP-9, and PAI-1, which are downstream targets of the TGF- signaling pathway; the expression of these genes accordingly showed.