[PMC free article] [PubMed] [Google Scholar] 12. P value was calculated from the raw data using Student’s t-test (P=0.001). C. Meta-analysis of mRNA levels in CC samples from the Oncomine database (http://www.oncomine.org). Box plots showing the increased expression of during tumorigenesis in CC datasets. 1: normal colon tissues, 2: normal rectum tissues, 3: cecum adenocarcinoma tissues, 4: rectal adenocarcinoma tissues, 5: colonadenocarcinoma tissues, 6: rectosigmoid adenocarcinoma tissues. The y-axis represents TRIM11 expression. Shaded boxes represent the interquartile range (25thC75th percentile). Whiskers represent the 10thC90th percentile. The bars denote the median. D. qRT-PCR analysis of TRIM11 mRNA levels cell lines. E. Western blot analysis of TRIM11 protein levels cell lines. F. CC patients with highTRIM11 expression exhibited significantly shorter overall survival OS and DFS compared with those with low expression, P 0.05. To investigate whether TRIM11 expression ARMD10 can serve as a novel prognostic marker for CC patients, based on the TRIM11 expression levels reported in a large public clinical microarray database, CC samples were subdivided into two groups and the associated overall survival (OS) and disease-free survival (DFS) were analyzed. Individuals with high TRIM11 levels exhibited shorter OS and DFS than those with low levels (Figure ?(Figure1F).1F). Collectively, these results indicate that TRIM11 is up-regulated in CC and that its high expression predicts a poor outcome for (+)-α-Tocopherol CC patients. Mir-24-3p down-regulation is responsible for TRIM11upregulation in CC cells To investigate how TRIM11 is up-regulated in CC cells, we first predicted which miRNAs regulated TRIM11 expression using TargetScan 5.1 (http://www.targetscan.org). Next, we selected 13 miRNAs with conserved binding to the 3UTR of TRIM11 mRNA in multiple species. These miRNAs were transfected into HCT116 cells, and endogenous TRIM11 protein was measured by Western blotting (Figure ?(Figure2A).2A). Meanwhile, these miRNAs were co-transfected with a reporter plasmid into HCT116 cells. pGL3-luc, which contains 13 miRNAs binding sites downstream of the luciferase gene, (+)-α-Tocopherol allows for quantitative measurement of TRIM11 3UTR activity. Figure ?Figure2A2A and ?and2B2B shows that miR-24-3p is the only miRNA that gave clear positive results in the two tests, indicating that miR-24-3p negatively regulates TRIM11 expression in CC cells. Importantly, mutation of the miR-24-3p seed region within the TRIM11 (+)-α-Tocopherol 3UTR abrogated the repressive ability of miR-24-3p (Figure ?(Figure2C2C and ?and2D),2D), demonstrating the specificity of the target sequence for TRIM11. Moreover, ectopic expression of miR-24-3p mimics can decrease TRIM11 mRNA level (Figure ?(Figure2E2E and ?and2F).2F). We asked whether this regulation extended to other CC cells; ectopic expression of miR-24-3p mimics also suppressed TRIM11 (+)-α-Tocopherol expression in SW480 and LoVo cells (Figure ?(Figure2G).2G). In contrast, TRIM11 protein levels increased after transfecting miR-24-3p inhibitors into DLD-1 and RKO cells (Figure ?(Figure2H).2H). These results indicate that miR-24-3p reduced the expression of TRIM11 through a direct seed sequence interaction. Open in a separate window Figure 2 TRIM11 is direct target of miR-24-3pA. Western blot analysis of TRIM11 protein levels after transfection of miRNAs mimics in HCT116 cells. B. Luciferase activity was measured 24 h after transfection of miRNAs mimics in 293T cells. Renilla luciferase was used for normalization. The bars correspond to the mean standard error, and the p-value was calculated using Student’s t-test. *P 0.05. C. The sequence of miR-24-3p and the 7-mer binding site in 3 UTR of TRIM11 mRNA. Red letters are the mutated nucleotides in the seed sequence of 3UTR. D. Mutant luciferase activity was measured 24 h after transfection of miR-24-3p mimics in 293T cells. E, F. The levels of miR-24-3p and TRIM11 were detected after transfection of miR24-3p mimics.2013;32:5038C5047. may be a useful new target for treating patients with CC. and levelspredict a poor outcomeA. qPCR analysis of TRIM11 expression in clinical CC samples of both tumor and the paired normal tissues. B. Meta-analysis of mRNA levels in CC samples from the MethHC database (http://methhc.mbc.nctu.edu.tw/php/index.php). Blue bars indicate mean value. The P value was calculated from the raw data using Student’s t-test (P=0.001). C. Meta-analysis of mRNA levels in CC samples from the Oncomine database (http://www.oncomine.org). Box plots showing the increased expression of during tumorigenesis in CC datasets. 1: normal colon tissues, 2: normal rectum tissues, 3: cecum adenocarcinoma tissues, 4: rectal adenocarcinoma tissues, 5: colonadenocarcinoma tissues, 6: rectosigmoid adenocarcinoma tissues. The y-axis represents TRIM11 expression. Shaded boxes represent the interquartile range (25thC75th percentile). Whiskers represent the 10thC90th percentile. The bars denote the median. D. qRT-PCR analysis of TRIM11 mRNA levels cell lines. E. Western blot analysis of TRIM11 protein levels cell lines. F. CC patients with highTRIM11 expression exhibited significantly shorter overall survival OS and DFS compared with those with low expression, P 0.05. To investigate whether TRIM11 expression can serve as a novel prognostic marker for CC patients, based on the TRIM11 expression levels reported in a large public clinical microarray database, CC samples were subdivided into two groups and the associated overall survival (OS) and disease-free survival (DFS) were analyzed. Individuals with high TRIM11 levels exhibited shorter OS and DFS than those with low levels (Figure ?(Figure1F).1F). Collectively, these results indicate that TRIM11 is up-regulated in CC and that its high expression predicts a poor outcome for CC patients. Mir-24-3p down-regulation is responsible for TRIM11upregulation in CC cells To investigate how Cut11 can be up-regulated in CC cells, we 1st expected which miRNAs controlled Cut11 manifestation using TargetScan 5.1 (http://www.targetscan.org). Next, we chosen 13 miRNAs with conserved binding towards the 3UTR of Cut11 mRNA in multiple varieties. These miRNAs had been transfected into HCT116 cells, and endogenous Cut11 proteins was assessed by Traditional western blotting (Shape ?(Figure2A).2A). In the meantime, these miRNAs had been co-transfected having a reporter plasmid into HCT116 cells. pGL3-luc, which consists of 13 miRNAs binding sites downstream from the luciferase gene, permits quantitative dimension of Cut11 3UTR activity. Shape ?Shape2A2A and ?and2B2B demonstrates miR-24-3p may be the just miRNA that gave crystal clear excellent results in both testing, indicating that miR-24-3p negatively regulates Cut11 manifestation in CC cells. Significantly, mutation from the miR-24-3p seed area within the (+)-α-Tocopherol Cut11 3UTR abrogated the repressive capability of miR-24-3p (Shape ?(Shape2C2C and ?and2D),2D), demonstrating the specificity of the prospective series for Cut11. Furthermore, ectopic manifestation of miR-24-3p mimics can lower Cut11 mRNA level (Shape ?(Shape2E2E and ?and2F).2F). We asked whether this rules extended to additional CC cells; ectopic manifestation of miR-24-3p mimics also suppressed Cut11 manifestation in SW480 and LoVo cells (Shape ?(Figure2G).2G). On the other hand, Cut11 protein amounts improved after transfecting miR-24-3p inhibitors into DLD-1 and RKO cells (Shape ?(Shape2H).2H). These outcomes indicate that miR-24-3p decreased the manifestation of Cut11 through a primary seed series interaction. Open up in another window Shape 2 Cut11 is immediate focus on of miR-24-3pA. Traditional western blot evaluation of Cut11 protein amounts after transfection of miRNAs mimics in HCT116 cells. B. Luciferase activity was assessed 24 h after transfection of miRNAs mimics in 293T cells. Renilla luciferase was useful for normalization. The pubs match the mean regular error, as well as the p-value was determined using Student’s t-test. *P 0.05. C. The series of miR-24-3p as well as the 7-mer binding site in 3 UTR of Cut11 mRNA. Crimson letters will be the mutated nucleotides in the seed series of 3UTR. D. Mutant luciferase activity was assessed 24 h after transfection of miR-24-3p mimics in 293T cells. E, F. The known degrees of miR-24-3p and TRIM11 were detected after transfection of miR24-3p mimics in HCT116 cells. G. Western.