The experimental protocol was approved by selleck kinase inhibitor the institutional ethics committee of the Yamaguchi University Graduate School of Medicine, Ube, Japan. The mean age of patients was 67��11 (mean��s.d.) years, and consisted of both men (70) and women (65). In all patients, the diagnosis of CRC was made on the basis of endoscopic and histological findings. A surgical operation was then carried out. The clinico-pathological characteristics after surgery are shown in Table 1. From the entire surgically resected tissue, viable tumour and non-tumour areas were macroscopically judged and cut out by pathologists. The tissues were immediately frozen in liquid nitrogen or subjected to RNA isolation, which were then stored at ?80��C. For RNA storage, RNAlater RNA Stabilization Reagent (Qiagen) was used.

Table 1 Characteristics of colorectal cancer patients Statistical analysis The relationship of FZD7 mRNA levels with clinical stage and follow-up information after surgery was analysed using the Kruskal�CWallis and post hoc tests. Kaplan�CMeier curves were compared using the log-rank test. Data were processed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Results Preparation and selection of FZD7_siRNA Thirteen shRNA expression vectors harbouring siRNAs against FZD7 were constructed and tested to determine which had the greatest suppressive effect on endogenous FZD7 expression in colon cancer cells. Each shRNA expression vector was transfected into HCT-116 cells and mRNA levels of FZD7 were examined by real-time PCR (Figure 1A).

The FZD7 expression in HCT-116 cells was reduced to 30% when using FZD7_siRNA8 compared to a control siRNA. Therefore we used FZD7_siRNA8 as an FZD7_siRNA for the following experiments. Figure 1 Preparation and selection of FZD7_siRNA. (A) Real-time PCR analysis of FZD7 mRNA expression in HCT-116 cells transfected with shRNA expression vectors harbouring siRNA against FZD7. Thirteen siRNAs were designed based on the nucleotide sequence of FZD7 … To examine whether the FZD7_siRNA could discriminate between FZD7 and FZD1 with the highest homology, we co-transfected FZD7-V5 or FZD1-V5 and FZD7_siRNA into 293T cells and subjected the whole proteins to immunoblotting with an anti-V5 antibody (Figure 1B). The expression of FZD7-V5 protein was abolished with FZD7_siRNA whereas that of FZD1-V5 was not.

Thus the FZD7_siRNA was used to specifically inhibit FZD7. FZD7_siRNA suppressed cell viability and invasion HCT-116 and HT-29 cells were transfected with scramble siRNA or FZD7_siRNA, and the cells were stained with crystal violet stain 6 days after transfection. Viable cells were decreased to <10 and 40% in HT-29 and HCT-116 cultures, respectively (Figure 2A). On the basis Anacetrapib of this result and the fact that we were unable to isolate stable HT-29 siRNA transfectants (see below), we used HCT-116 cells for the following siRNA transfection experiments.