and S.L. analyzed by quantitative real-time reverse transcription PCR (RT-PCR). Western blot analyses confirmed that SLP-2 protein expression was higher in tumors than in paired adjacent nontumor tissues (Figure?1B). Furthermore, the immunohistochemical (IHC) staining of SLP-2 was performed in colorectal adenomas (n?= 50), high-grade intraepithelial neoplasias (HGINs; n?= 50), invasive carcinoma, and paired adjacent nontumor tissues (n?= 491), and we GSK726701A found that the proportion of tumors with high SLP-2 expression progressively increased when nontumor tissues progressed to invasive carcinoma (Figure?1C), which suggests that SLP-2 may be associated with tumor progression. The localization of GSK726701A SLP-2 expression was cytoplasmic, and representative staining showed the negative, weak, GSK726701A moderate, and strong expressions of SLP-2 in nontumor tissue, adenoma, HGIN, and invasive carcinoma, respectively (Figure?1D). Tumors with high SLP-2 expression (n?= 223) were associated with clinicopathological features that were indicative of a more aggressive phenotype, which included the depth of tumor invasion, lymphatic and/or venous invasion, nodal involvement, distant metastasis, and tumor, node, metastasis (TNM) staging (Table S3). As shown in Figures 1E and 1F, a significantly higher proportion of poorly differentiated CRC than of highly and GSK726701A moderately differentiated invasive carcinomas displayed increased SLP-2 staining intensity. No significant difference was found regarding patient age, gender, carcinoembryonic antigen (CEA) levels, and histological type between tumors with low and high SLP-2 expression (Table S3). Additionally, log-rank analyses revealed that patients with high SLP-2 expression had significantly shorter overall survival (OS) and progression-free survival (PFS) (p?< 0.0001; Figures 1G and 1H) than patients with low SLP-2 expression. Multivariate Cox regression analyses further confirmed that high SLP-2 expression, similar to other prognostic factors, such as age, distant metastasis, and TNM stage, was an independent prognostic factor for CRC (hazard ratio, 0.469; p?= 0.006) (Table S4). Open in a separate window Figure?1 High SLP-2 Expression Correlates with Tumor Progression and Poor Prognosis in CRC (A) Quantitative assessment of the transcript in 74 CRC and matched adjacent normal tissue samples. (B) Representative western blot analysis of SLP-2 protein levels in five paired CRC and matched adjacent normal tissue samples. (C) Stacked bar plots showing the percentage of patients with high or low SLP-2 protein expression in colorectal adenoma, HGIN, invasive carcinoma, and matched adjacent nontumor tissue samples. (D) Representative micrographs of SLP-2 protein expression in nontumor, adenoma, HGIN, and invasive carcinoma (scale bars, 100?m). (E) Representative micrographs of SLP-2 protein expression in highly, moderately and poorly differentiated CRC (scale bars, 50?m). (F) Stacked bar plots indicating the number of patients with high or low SLP-2 protein expression that had highly, moderately, and poorly differentiated CRC. (G and H) OS (G) and PFS (H) of CRC patients with low (red line) or high (blue line) SLP-2 expression. Error bars represent the mean??SEM. ?p?< 0.05, ???p?< 0.001, two-tailed, Rabbit polyclonal to ZBED5 unpaired t tests. N & Non-T, nontumor tissue; T, tumor; Ade, adenomas; HGIN, high-grade intraepithelial neoplasia; Ica, invasive carcinoma; Dif, differentiation; PFS, progression-free survival; OS, overall survival. SLP-2KO Arrests CRC Cell Growth and findings, SLP-2KO significantly inhibited CRC xenograft growth compared to that of the control cells (Figure?2F). Additionally, all tumors from the two groups were dissected, fixed, and stained with hematoxylin and eosin (H&E) and the proliferation index Ki-67. Consistent with the findings in CRC clinical samples, morphological analysis suggested that SLP-2 was correlated with tumor differentiation, as SLP-2KO induced adenoid differentiation in samples from HCT116and and in isogenic cells and found that SLP-2KO significantly downregulated the mRNA levels of and and mRNA expression in HCT116 and SW480 cells that were transduced with SLP-2KO-sg1 and -2. Data are presented.