Sample collection and extraction of RNA
A total of 41 colorectal tumor samples resected by ESD (endoscopic submucosal dissection) or EMR (endoscopic mucosal dissection) from September 2011 to June 2013 at the Chiba University Hospital were selected for this study. The morphological classification and original pathological diagnoses were 17 LST-adenoma, 12 LST-carcinoma, and 12 Ip-adenoma, all 10 mm or more in diameter. Tumor and normal mucosa tissues were taken from the same patient during endoscopic treatment and incubated in RNA later® (Ambion, Inc., Austin, Texas USA) for 48 h at 4 °C, and then stored at −80 °C. Total RNA was extracted using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) according to the manufacturer’s instructions and stored at −80 °C until further use. This study was approved by the ethics committee of Chiba University and written informed consent was obtained from all patients (UMIN 000009998).
PCR array analysis
RNA samples were reversed transcribed using RT2 First Strand Kits (SA Biosciences Corp., Frederick, MD, USA). The resulting cDNA was analyzed using the Human Cancer Pathway and Human Extracellular Matrix & Adhesion Molecules RT2 Profiler™ PCR Arrays (SA Biosciences Corp.), which comprises a panel of 84 primer sets related to genes involved in the cytoskeleton, cell proliferation, cell-cell interactions and cell-matrix interactions. We examined 168 genes in total.
All results were normalized relative to expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR arrays were analyzed using the 7300RT-PCR System (AB Applied Biosciences). The amplification protocol consisted of an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. The expression of each gene was quantified based on its Ct (cycle threshold), the number of cycles at which the linear phase crossed the threshold level.
Gene expression data analysis
Statistical analysis of the PCR array data was performed using an online analysis tool (SA Biosciences Corp., http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php), with statistical significance set at p ≤ 0.05. Relative changes in gene expression on quantitative RT-PCR were analyzed using the ΔCt or ΔΔCt method. Data were normalized by subtracting the average Ct value of the reference gene (GAPDH) from that of the sample genes in each group (17 LST-adenoma, 12 LST-carcinoma, 12 Ip-adenoma), with the normalized Ct value designated the ΔCt value for that gene. ΔCt was compared to control samples using the equation ΔΔCt = (ΔCt sample –ΔCt control) and fold changes in target gene expression were calculated using the formula, expression fold change = 2(−ΔΔCt).
Hierarchical and K-means clustering analysis
To demonstrate statistical significant difference in gene expression between LSTs and polypoid tumors, based on the results of PCR array analysis, we performed comparative statistical analyses, such as hierarchical and K-means clustering analysis, using GeneSpring GX 12.0 (Agilent) software (Chemical Evaluation and Research Institute, Saitama, Japan). Expression levels of 129 genes (the remaining 39 genes did not yield statistically efficient results in the PCR array analysis) in the 41 colorectal tumor samples were analyzed by hierarchical clustering. Based on these results, we performed K-means clustering to clarify the nature of gene expression in LSTs.
Immunohistochemistry
To confirm the gene expression, immunohistochemical analysis was performed on 60 additional samples obtained from ESD or EMR for colorectal tumors from May 2003 to April 2013 at the Chiba University Hospital. These comprised 38 LST-adenoma and 22 Ip-adenoma samples. Sections of colorectal tumors resected by ESD or EMR were fixed and stained with hematoxylin-eosin for histological examination. We used the following antibodies: TNFRSF25, AKT1, BCL2L1, MTA2 and ERBB2 (1:200, Abcam plc., Cambridge, UK). Positively stained cells were visualized using 3’3-diaminobenzidine and counterstained with Mayer’s hematoxylin. Immunoreactivity was independently evaluated by two researchers who were blinded to patient outcome and pathology. We took the brown-colored cytoplasm cells as positive expressions. When more than half of cells showed the immunoreactive stains in one high power field, we judged as “positive” in this field. Then we selected nine high power fields randomly and judged each field respectively. If “positive” in more than half of fields was detected, we ultimately judged as “positive”. When there was any discrepancy in their evaluations, the samples were reexamined by both researchers.
Evaluation of Apoptosis status in LST-Adenoma
To evaluate the apoptosis status in LST-adenoma, 38 LST-adenoma samples were analyzed by the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine-triphosphate nick-end labeling (TUNEL) method. Apoptotic cell were identified by In situ Apoptosis Detection Kit (Takara Bio Inc., Shiga, Japan). TUNEL positivity was indicated by homogeneously brown nuclear staining. The apoptotic index was defined as the number of TUNEL-positive cells in randomly selected nine high power fields. When the apoptotic index was more than nine, we ultimately judged as “positive”.
Statistical analysis
All results are expressed as means ± SD. Statistical differences between two groups were determined by Student’s t-test using SPSS 16.0 J (SPSS Inc., Chicago,IL, USA), with p < 0.05 considered statistically significant.