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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.
Colon cancer (CC) is the third most common cancer and the fourth leading cause of cancer-related deaths worldwide [1–3]. Mortality remains high despite improvements in prevention and treatment. CC, therefore, remains a major health problem. The dysregulation of oncogenes or tumor suppressor genes is tightly correlated with CC initiation, progression, and resistance to therapy, of all of which involve changes in the biological characteristics of cancer cells, including cell growth, apoptosis, migration, invasion, and metabolism [4, 5]. The identification of novel therapeutic targets, less toxic therapies, and better predictive markers is urgently needed.
TRIMs, members of the RING family of Ub E3 ligases, are characterized by the presence of three conserved domains, RING, B-Box, and coiled-coil (RBCC) . TRIM family proteins are involved in many biological processes, and changes in their abundance or activity are associated with several pathological conditions, including viral infections, developmental and neurodegenerative disorders, and cancers [7, 8]. TRIM11 binds to and destabilizes Humanin, an inhibitor of Alzheimer-like neuronal insults . TRIM11 destabilization of the activator-mediated cofactor complex (ARC105) suppresses ARC105-mediated transcriptional activation induced by transforming growth factor β signaling . Trim11 promotes Pax6 degradation, and the subsequent inhibition of Pax6 transcriptional activity impairs neurogenesis . TRIM11 interacts with Phox2b, a homeodomain transcription factor that modulates the development of noradrenergic neurons, and increases expression of dopamine β-hydroxylase gene . In addition, TRIM11 is upregulated in malignant gliomas, where it promotes proliferation, invasion, migration, and tumor growth by increasing the accumulation of EGFR and activity of MAPK cascade . TRIM11 is also highly expressed in lung cancer tissues and cell lines, and higher expression of TRIM11 is correlated with the poorer prognosis in lung cancer patients .
MicroRNAs (miRNAs) are non-coding, single-stranded RNA molecules (~22 nucleotides in length), which post-transcriptionally regulate gene expression by binding to the 3’-untranslated region (3’-UTR) of specific mRNAs and targeting them for degradation or translational repression [15–17]. Among these microRNAs, miR-24-3p has been shown to act as a cell type specific oncogene or tumor suppressor in a variety of cancers [18–20]. Gao et al.  demonstrated that miR-24-3p was downregulated in human CC tissues relative to corresponding non-cancerous tissues, and overexpression of miR-24-3p suppressed CC cell proliferation, migration, and invasion in vitro. Mishra et al.  demonstrated that miR-24 functions as a tumor suppressor independent of p53 by targeting and repressing dihydrofolate reductase in CC cell lines. In addition, Fang et al.  showed that the plasma levels of miR-24 were decreased in patients with CC and benign lesions (polyps and adenoma) compared with healthy controls, revealing miR-24as a promising potential biomarker for CC detection. However, little is known about the targets of miR-24-3p in CC.
In this study, we evaluated the expression of TRIM11 in clinical CC tissues compared and adjacent non-cancerous tissues, and examined the relationship between TRIM11 expression and clinical outcomes. We also sought to evaluate the effect of TRIM11 expression on CC cell phenotypes in vitro and in vivo. Moreover, we examined miRNAs targeting TRIM11 in order to shed light on the mechanisms of TRIM11 regulation and dysregulation in CC.
We measured the mRNA level of TRIM11 in CC tissues and normal colon tissues using quantitative PCR (qPCR). As shown in Figure Figure1A1A and Supplementary Figure 1, the TRIM11 mRNA level was significantly higher in 22 out of 23 tumor tissues than in the paired normal colon tissues. We analyzed its expression using online databases , as shown in Figure Figure1B1B and and1C,1C, and found TRIM11 is significantly up-regulated in CC tissues compared with the normal tissues (P<0.01). Likewise, we found that TRIM11 mRNA and protein levels were elevated in all of the ten CC cell lines examined, relative tothe normal colon fibroblast cell line (CCD18-Co) (Figure (Figure1D1D and and1E),1E), suggesting that TRIM11 is up-regulated in CC and may be related to CC progression.
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.
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 3’UTR 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, allows for quantitative measurement of TRIM11 3’UTR 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 3’UTR 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 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.
To further verify the relationship between TRIM11 and miR-24-3p, we detected miR-24-3p expression level by qRT-PCR in the 23 pairs of CC and non-tumor colon tissues, in which TRIM11 expression level had been measured. miR-24-3p was downregulated in tumor tissues compared with normal colon tissues (Figure (Figure3A)3A) and TRIM11 was negatively correlated with miR-24-3p expression level (Pearson r = -0.32, P = 0.028) (Figure (Figure3B),3B), which suggested that the upregulated TRIM11 was, at least in part, due to downregulated miR-24-3p in CC.
We stably overexpressed TRIM11 in CC cell lines (Figure (Figure4A).4A). During cell culture, we noticed that TRIM11-overexpressing cells proliferated faster than control counterparts. Accordingly, a significant increase in the proliferation of TRIM11 overexpressing cells compared to control cells was observed by cell counting kit-8 (CCK-8) assay (Figure (Figure4B4B and and4C).4C). Colony formation assay also indicated that overexpression of TRIM11 significantly promoted cell colony formation ability (Figure (Figure4D4D and and4E4E).
Induction of apoptosis is a therapeutic strategy for CC treatment. To address whether TRIM11 regulated CC cell apoptosis, the propidium iodide-annexin V assay was performed; after treating DLD-1 cells with 5-FU for 24 hours, 15% of control cells underwent apoptosis, while the rate among TRIM11-expressing cells was 10% (Figure (Figure4F4F and and4G).4G). Taken together, these findings indicated that overexpression of TRIM11 promotes cell proliferation and inhibits apoptosis in CC.
The ability to knock out genes by CRISPR/Cas9 mediated genome editing is revolutionizing modern genetics . We constructed three sgRNAs targeting different regions in one of the first few exons of the human TRIM11 gene, using bioinformatics prediction to avoid obvious potential off-target effects . Western blotting showed that only sgRNA#2(KD2) efficiently knocked down TRIM11 in HT29 and HCT116 cells (Figure (Figure4A).4A). During cell culture, we noticed that KD2 cells proliferated more slowly than the control counterpart. Furthermore, CCK-8 assay indicated that silencing TRIM11 significantly suppressed cell proliferation (Figure (Figure5A5A and and5B).5B). The colony formation assay showed that silencing TRIM11 decreased colony formation ability (Figure (Figure5C5C and and5D).5D). Furthermore, the propidium iodide-annexin V assay revealed that knockdown of TRIM11 increased the apoptosis rate after treating with 5-FU for 24 hours (Figure (Figure5E5E and and5F).5F). Collectively, our result suggests that reduced TRIM11 suppresses CC cell proliferation and induces apoptosis in vitro.
To test whether knocking down TRIM11 suppressed CC tumor growth in vivo, we inoculated nude mice with HT29 and its derived cells. After 4 weeks, the tumors formed by the control cells were larger and heavier than the TRIM11-silenced tumors, (Figure 6A-6C). Together, these findings indicated that silencing TRIM11 decreased tumor growth in vivo.
A better understanding of the mechanisms underlying CC development, progression, and therapy resistance is urgently needed to guide the design of novel effective therapies for this deadly cancer. TRIM11 expression is upregulated, and it functions as an oncogenic protein in malignant gliomas  and lung cancer . In this report, we demonstrate for the first time that TRIM11 is a key player in CC progression; we also show that downregulation of miR-24-3p is at least partly responsible for the upregulation of TRIM11 in CC cells. This novel miR-24-3p/TRIM11 axis may be useful for the development of new strategies for treating patients with CC.
We demonstrated that the mRNA and protein levels of TRIM11 were significantly increased in CC by performing data mining, qPCR analysis, and western blotting. High TRM11 expression was associated with poor outcomes in patients with CC, indicating that the high level of TRIM11 was likely to present prognostic value. To explore the mechanism underlying the increased expression of TRIM11 in CC cells, we showed that miR-24-3p was at least one of the negative regulators of TRIM11. Gao Y et.al  showed that miR-24-3p was downregulated in human CC and was a prognostic factor for OS and DFS of CC patients. Transfection of miR-24-3p mimics significantly decreased cell number in SW480 and HT29 cells . Our data also concurred with previous studies that miR-24-3p could inhibit cell proliferation in CC. Our data demonstrate that miR-24-3p directly targets TRIM11, suppressing cell proliferation, which suggests that miR-24-3p may play a suppressor role by targeting TRIM11.
E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many proteins for targeted degradation by the 26S proteasome and play a key role in carcinogenesis . E3 ubiquitin ligases are important regulators of a variety of biological processes including cell cycle regulation, cell proliferation, and apoptosis [27, 28]. The TRIM family of genes has been widely studied due to their key roles in development, differentiation and host cell antiviral defenses; however, roles in cancer biology are emerging . For example, TRIM24 is an oncogenic transcriptional activator in prostate cancer . TRIM15 expression is decreased in human colon adenocarcinoma compared with normal colon tissues; restoring expression in CC cells suppressed tumor growth in mice . Our data shows that overexpression of TRIM11 promoted CC cell proliferation and inhibited apoptosis; in contrast, knockdown of TRIM11 using CRISPR/Cas9-mediated gene editing technology suppressed cell proliferation and induced apoptosis. Furthermore, knockdown of TRIM11 suppressed tumor growth in vivo. Those data indicated that TRIM11 functions an oncogene in CC.
In summary, our findings demonstrate that TRIM11 plays an oncogenic role and could serve as a clinical predictor in CC. We also highlight that TRIM11 expression dysregulation involves the miR-24-3p-mediated machinery, all of which offer potential avenues for the treatment of this fatal disease. Together, these data provide new insights into the molecular basis of this deadly malignancy.
Nine human colorectal cancer cell lines (HCT116, HT29, SW480, SW620, DLD-1, LoVo, HCT8, RKO, and CaCo2) and the human colon fibroblast cell line CCD18-Co were purchased from the American Type Culture Collection (ATCC) and cultured according to their instructions. All cell lines used in this study were authenticated through short tandem repeat profiling less than 6 months ago when this project was initiated, and the cells have not been in culture for more than 2 months.
The full-length cDNA of human TRIM11 was cloned into the pSin-puro vector (Focus Bioscience Co., Ltd, Nanchang, China). sgRNA-NC targeting EGFP (with no known targets in the human genome) was cloned intothe lentiCRISPR V1 vector; sgRNA-NC: GGGCGAGGAGCTGTTCACCG. TRIM11 single guide RNA (sgRNA) plasmid ligated into the lentiCRISPR V1 vector (Focus Bioscience Co., Ltd, Nanchang, China); sgRNA#1: 5’-ACGCATCCTGCATCTGCTTC-3’; sgRNA#2: 5’-TGCGTTGCTGTTCCAAGCCC-3’; sgRNA#3: 5’-CGGATGAGACCTGCG TCTTG-3’. The three sgRNAs targeting the exons of TRIM11 gene were selected from a published database of predicted high-specificity protospacer-adjacent motif target sites in the human exome . The full-length 3′UTR of TRIM11 was cloned by standard procedures into the pGL3.0-control vector (Promega), immediately downstream of the stop codon of the luciferase gene to generate the TRIM11-Luciferase-WT luciferase reporter plasmid (Focus Bioscience Co., Ltd, Nanchang, China). Mutagenesis of the TRIM11-Luciferase-WT was performed using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA).
Anti-GAPDH was procured from Nanchang Focus Bioscience Co., Ltd. Anti-TRIM11 (HPA001209) was obtained from Sigma-Aldrich.
pSin-puro-TRIM11, pSin-puro-empty vector, lentiCRISPR-NC, lentiCRISPR-sgRNA#1, lentiCRISPR-sgRNA#2 or lentiCRISPR-sgRNA#3 was co-transfected with pMD.2G and psPAX2 into HEK-293T cells for 48 h. The recombinant viruses were subsequently collected and added to CRC cells cultured with 8 μg/ml polybrene for 24 h. The stable lines were selected with 1μg/ml puromycin for two weeks (Focus Bioscience Co., Ltd, Nanchang, China).
These procedures were performed as previously described . Briefly, total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized using Revert Aid™ First Strand cDNA Synthesis Kit (MBI Fermentas). The primers employed for amplifying TRIM11 and GAPDH were validated. TRIM11 primers are as follows: GAPDH primers are as follows:F 5’-ACAGTCAGCCGCATCTTCTT-3’ and R 5’-GACAAGCTTCCCGTTCTCAG-3’. TRIM11 primers are as follows:F 5’-GAGAACGTGAACAG GAAGGAG-3’ and R 5’-CCATCGGTGGCACTGTA GAA-3’. The expression of miR-24-3p was quantitated in human tissues using the mirVana qRT-PCR miRNA Detection Kit and the miR-24-3p and U6 snRNA primer sets (Ambion) in a Roche LightCycler (Roche, Basel, Switzerland).
In vitro cell proliferation was assessed using the CCK-8 assay; cells were seeded in 96-well plates at a density of 1,000 cells/well and incubated for 1, 2, 3, 4, or 5 days. Ten microliters of the CCK-8 reagent (Cell Counting Kit-8, Beyotime, China) was then added to each well, followed by incubation for 1.5 h. The absorbance value (OD) of each well was measured at 450 nm. For each experimental condition, 6 wells were used.
The miRNA mimics (miR-24-3p, miR-101-3p, miR-148a, miR-15a, miR-504, miR-329, miR-370, miR-149, miR-320a, miR-193b, miR-544a, miR-150, miR-124-3p), miRNA control, and miRNA inhibitors for miR-24-3p were purchased from Ribobio (Ribobio, Guangzhou, China). After seeding into 6-well plates, cells were transfected with miRNA mimics at a final concentration of 100 nM using RNAiMAX Reagent (Invitrogen).
Cells were plated in 6-well culture plates at 250 cells per well. Each group included 3 wells. After incubation for 15 days at 37°C, the cells were washed twice with PBS and stained with Giemsa solution (Focus Bioscience Co., Ltd, Nanchang, China). The number of colonies containing ≥50 cells was counted under a microscope.
Apoptosis analysis was conducted with an Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, China) according to the manufacturer's protocol. The percentage of apoptotic cells was determined using FACS flow cytometry and associated software (BECKMAN).
Kaplan-Meier survival curves for TRIM11 were obtained using the tools at http://www.canevolve.org based on the GSE17536 data set, probe set 226566_at.
This process was carried out as described previously [33, 35]. Briefly, the cells were plated in 12-well plates at the density of 2 × 105 per well, and were transfected with 0.8 μg of luciferase plasmid. To normalize transfection efficiency, the cells were cotransfected with 8 ng of pRL-CMV (Renilla luciferase). After transfection for 48 hrs, luciferase activity was measured using the Dual-Luciferase Assay kit (Promega). Three independent experiments were performed, and their calculated means and standard deviations are presented.
The use of human CC tissues was reviewed and approved by the ethics committee of Jiangxi Cancer Hospital and was performedin accordance with approved guidelines. Informed consent was obtained.
All statistical analyses were performed using SPSS for Windows, version 16.0(SPSS). Pearson's correlation analysis was performed to assess the relationships between miR-24-3p and TRIM11 in the tissues using mRNA expression data from qRT-PCR. All values from the in vitro assays are expressed as the mean±SD or SEM of at least three independent experiments or replicates. P values were calculated using the two-tailed Student's test. A p-value< 0.05 is considered statistically significant.
This work was supported by the National Nature Science Foundation of China (NSFC) (Grant No. 81260337 to LL; Grant No. 81660414 to LL; Grant No. 81660449 to YS; Grant No. 81460613 to JZL; Grant No. 81460430 to SWL), Jiangxi Provincial Natural Science Foundation of China (Grant No. 20161ACB21001, to YS).
CONFLICTS OF INTEREST
The authors have no potential competing interests as defined by Oncotarget, or other interests that might be perceived to influence the results and/or discussion reported in this article.