TRIM11, a direct target of miR-24-3p, promotes cell proliferation and inhibits apoptosis in colon cancer

Abstract

TRIM11 (tripartite motif-containing protein 11) is an E3 ubiquitin ligase recently identified as an oncogene in malignant glioma and lung cancer. In the present study, we report that expression of TRIM11 was increased in colon cancer (CC) tissue relative to paired normal tissues and that higher TRIM11 levels predicted poor overall survival (OS) and disease-free survival (DFS) in CC patients. Mechanistically, we showed that miR-24-3p downregulation contributes to TRIM11 upregulation in CC. We also demonstrated that TRIM11 overexpression promotes cell proliferation and colony formation and inhibits apoptosis in CC, while knocking down TRIM11 using CRISPR/Cas9-mediated genome editing inhibited cell proliferation and induced apoptosis. Silencing TRIM11 in vivo decreased tumor growth. These findings indicate that TRIM11 facilitates CC progression by promoting cell proliferation and inhibiting apoptosis and that the novel miR-24-3p/TRIM11 axis may be a useful new target for treating patients with CC.

Keywords: TRIM11; colon cancer; miR-24-3p.

Figure 1. TRIM11 expression is increased in CC, and higher TRIM11 levelspredict a poor outcome

A. qPCR analysis of TRIM11 expression in clinical CC samples of both tumor and the paired normal tissues. B. Meta-analysis of TRIM11 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 TRIM11 mRNA levels in CC samples from the Oncomine database (http://www.oncomine.org). Box plots showing the increased expression of TRIM11 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 (25^th–75^th percentile). Whiskers represent the 10^th–90^th 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.

Figure 2. TRIM11 is direct target of miR-24-3p

A. 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 3’UTR. 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 in HCT116 cells. G. Western blot analysis of TRIM11 protein levels after transfection of miR24-3p mimics. H. Western blot analysis of TRIM11 protein levels after transfection of miR24-3p inhibitors.

Figure 3. TRIM11 was negatively correlated with miR-24-3p in CC

A. The miR-24-3p level was detected by qRT-PCR in 23 pairs of CC and corresponding non-tumor colon tissues. B. Pearson's correlation between miR-24-3p and TRIM11 expression levels was analyzed, showing a significant negative correlation (r = -0.32, P = 0.028).

Figure 4. Overexpression of TRIM11 promotes CC cell proliferation and inhibits cell apoptosis

A. The generation of stable cell lines in which TRIM11 was overexpressed or silenced was confirmed by western blotting. GAPDH was used as the internal control. B, C. The cell proliferation of the indicated stable cell lines in vitro was measured at different time points, as indicated by the CCK-8 assay. The bars correspond to the mean± standard error, and the P value was calculated using Student's t-test. *P<0.05, **P<0.01. D, E. The colony formation of the indicated stable cell lines in vitro was measured for 14 days. The bars correspond to the mean± standard error, and the p-value was calculated using Student's t-test. **P<0.01,***P<0.001. F, G. The stable cell lines overexpressing the empty vector or TRIM11 were treated with 150 μg/ml5-FU for 24 h and then subjected to annexin V-FITC and PI staining. Cell apoptosis was evaluated through FACS. The bars correspond to the mean± standard error (n = 3), and the P-value was calculated using Student's t-test. *P<0.05.

Figure 5. Knockdown of TRIM11 suppressed CC cell proliferation and induced apoptosis

A, B. The cell proliferation of the indicated stable cell lines in vitro was measured at different time points, as indicated by the CCK-8 assay. The bars correspond to the mean± standard error, and the P value was calculated using Student's t-test. *P<0.05, **P<0.01. C, D. The colony formation of the indicated stable cell lines in vitro was measured for 14 days, as described in the Methods. The bars correspond to the mean± standard error, and the p-value was calculated using Student's t-test. **P<0.01,***P<0.001. E, F. The stable cell lines silencing the negative control or TRIM11 were treated with 150 μg/ml5-FU for 24 h and then subjected to annexin V-FITC and PI staining. Cell apoptosis was evaluated through FACS. The bars correspond to the mean± standard error (n = 3), and the p-value was calculated using Student's t-test. *P<0.05.

Figure 6. Silencing TRIM11 reduces tumor growth in vivo

A. Tumor weights for the tumors formed by the indicated cells (p=0.0046). B. Representative images of the tumors from all the mice in each group. C. H&E staining representative images of tumor formed by KD2 and NC cells.