Histological Analysis selleck inhibitor For pathology analysis, 4-μm thick sections of formalin-fixed, paraffin-embedded tissues were prepared. After hematoxylin and eosin staining, the sections of each tumor were examined under a light microscope (Olympus, Japan). RNA extraction and Real-time polymerase chain reaction labeling, hybridization, and analysis Total RNAs from normal colonic mucosa of all groups were got using TRIzol (Invitrogen, USA) according to manufacturer’s instruction. RNA content and purity were measured using Nanodrop ND-1000, and denaturing gel electrophoresis was performed. Next, Reverse transcription and quantification of gene expression was performed according to the
manufacture’s introduction (Takara). We used 18s as an internal control in Real- time PCR. Next, 3 samples of non-tumor colon of the group of NS, DMH, FA2, FA3 were amplified and labeled with the Agilent Quick Amp labeling kit and hybridized using Agilent whole genome oligo microarray (Agilent Technologies, Palo Alto, CA, USA) by using Agilent SureHyb Hybridization Chambers. Then, the processed slides were scanned with the Agilent DNA microarray scanner according to the settings provided by Agilent Technologies. The microarray data sets were normalized by Agilent GeneSpring
Tariquidar cell line GX software (version 11.0) using the Agilent FE one-color scenario (AZD8931 mainly median normalization). Differentially expressed genes were identified via the fold-change (FC) and p values of the t-test. Differentially expressed genes are identified to have an FC of ≥ 1.5 and a p value of ≤ 0.05 between two groups. Functional differences of the differentially expressed genes was analyzed using the Gene Ontology (GO; http://www.geneontology.gov/). Statistical analysis The results of the animal experiments and real-time PCR were analyzed
using SAS 9.2 software (SAS Institute Inc. USA) with data presented in the forms of means ± SD. Student’s t-test was used to compare values between two independent groups. Differences were considered to be significance when p < 0.05. Results Results of Animal Experiment In the 12th week, 2 of 20 mice in DMH group PTK6 were discovered average 2 × 3 mm adenoma, while there is none in FA1 and NS groups. Thus, the 12th week after DMH treatment might be considered to be the pre-stage that adenomas formed in DMH-induced model. We have successfully induced CRC in the animal model with injection DMH for 24 weeks, which were identified as adenocarcinoma by histology analysis (Figure 2A, B). Figure 1 shows mainly results of the experiment. We can see that the incidence of DMH-induced group is 90%, much higher than any other groups such as FA2, FA3, which are 63%, 45% respectively (Figure 2C). There is significant difference between groups of FA3 and DMH but not between FA2 and DMH groups.