Repared MKN74 cells with distinct CRKL expression levels using the siRNA
Repared MKN74 cells with distinct CRKL expression levels using the siRNA knockdown of CRKL expression. CRKL-specific siRNA transfection effectively decreased the level of CRKL protein expression in MKN74 cells by approximately 70 of the levels observed in LCZ696 chemical information negative control siRNA-transfected cells (Figure 2A). A cell proliferation assay showed that the number of CRKL siRNA-transfected MKN74 cellswas significantly lower at 3 and 4 days after transfection than the number of negative control siRNA-transfected cells (Figure 2B), meaning that CRKL has the ability to upregulate cell proliferation.Overexpression of CRKL protein in gastric cancerTable 1 Detection of chromosomal regions with a high copy number (more than 6) in the gastric cancer cell lines MKN7, MKN28, and MKN74 using a genome-wide SNP microarray analysisChromosomal regionsa 9p13 17q12-q21 19q12 19q13 22q11 Genes with a high copy number in the region PAX5 FBXL20, MED1, PERLD1, ERBB2, IKZF3, ZPBP2 CCNE1 CD22 DGCR8, USP41, ZNF74, SCARF2, KLHL22, MED15, PI4KA, SERPIND1, SNAP29, CRKL, THAP7, P2RX6, LOCa If more than four consecutive SNP probes with a copy number of more than six were detected in either of the three cell lines, the chromosomal region was regarded as being a “highly amplified region” and was listed in this table.Next, we investigated the expression status of CRKL protein in primary gastric cancer using an immunohistochemical analysis with anti-CRKL monoclonal antibody (Y243). CRKL was mainly observed in the cytoplasm, consistent with previous reports [29]. When we compared the level of CRKL expression between non-cancerous gastric foveolar epithelium (n = 41) and gastric cancer (n = 360), the level of CRKL expression in gastric cancer (mean ?standard deviation = 0.42 ?0.63) was significantly higher PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 than that in non-cancerous tissue (0.20 ?0.26) (P = 0.032) (Figures 3A-3E). When an expression level of 1.00, which corresponds to a value 5-fold of the mean expression level in non-cancerous gastric foveolar epithelium, was used as a cutoff value for the expression status in gastric cancer (i.e., low expression group, 0?.99; high expression group, 1.00?.00), 88 (24.4 ) of the 360 primary gastric cancers were included in the high expression group (Figure 3F). To examine whether CRKL overexpression is associated with CRKL amplification in gastric cancer, we performed a FISH analysis for the CRKL gene in the 360 primary gastric cancers and compared the prevalence of CRKL amplification between the low expression group and the high expression group. AsNatsume et al. Journal of Translational Medicine 2012, 10:97 http://www.translational-medicine.com/content/10/1/Page 8 ofexpected, the percentage of gastric cancer cells with CRKL amplification was significantly higher in the high expression group (9.1 ; 8/88 cases) than in the low expression group (2.2 ; 6/272 cases) (P = 0.028, chi-square test). This result suggests that CRKL amplification contributes to CRKL overexpression in primary gastric cancer. We further investigated whether the levels of CRKL expression is associated with clinicopathological features in primary gastric cancer patients, the high CRKL expression was observed significantly more often in male and differentiated-type gastric cancer (Table 2). These results suggested that CRKL protein is overexpressed partly due to CRKL amplification in a subset of primary gastric cancers and is associated with the gender and histopathology.Decrease in the viability of CRKL-exp.
