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. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Gastroenterology. 2015 Jun 6;149(4):981–992.e11. doi: 10.1053/j.gastro.2015.05.057

MicroRNA214 is Associated with Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-associated Cancer in Mice

Christos Polytarchou 1, Daniel W Hommes 2,3,*, Tiziana Palumbo 1,*, Maria Hatziapostolou 1,*, Marina Koutsioumpa 1, Georgios Koukos 1, Andrea E van der Meulen-de Jong 2, Angelos Oikonomopoulos 1,3, Welmoed K van Deen 2,3, Christina Vorvis 1, Oksana B Serebrennikova 4, Eleni Birli 1, Jennifer Choi 3, Lin Chang 5, Peter A Anton 6, Philip N Tsichlis 4, Charalabos Pothoulakis 3, Hein W Verspaget 2, Dimitrios Iliopoulos 1,#
PMCID: PMC4584179  NIHMSID: NIHMS698191  PMID: 26055138

Abstract

Background & Aims

Persistent activation of the inflammatory response contributes to development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC).

Methods

We performed quantititave PCR analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice were also given inhibitor of microRNA214 (miR214).

Results

A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor κB (NFκB). Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses revealed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced STAT3-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsies from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN.

Conclusions

Interleukin-6 upregulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates NFκB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

Keywords: IL6, IBD progression, mouse model, chronic inflammation

Introduction

There is an increasing incidence of UC potentially due to the westernized way of life, diet and environmental alterations1, 2, while patients with long standing and extensive disease are at increased risk of developing colorectal cancer3-5. Although early studies have shown standardized incidence ratios between 3.7% and 5.7%,3, 4 the magnitude of this risk is unknown. More recent studies report much lower rates most likely related to improved therapeutic management.5-9 Practice guidelines recommend lifelong surveillance colonoscopies at intervals of 1 to 3 years, starting 8 to 10 years after diagnosis.10 The rationale of surveillance colonoscopy is to detect premalignant mucosal dysplasia. Patients with high grade dysplastic lesions are recommended to undergo prophylactic proctocolectomy, while for lesions of low grade dysplasia there is insufficient evidence to recommend surgery.10 Medical decision analysis failed to show benefit of surveillance colonoscopy, and presently, there are also no clinical data to support the contention that surveillance colonoscopy is a cost effective means to prevent death from colorectal cancer in UC patients.11 Improving surveillance strategies with the use of molecular markers has not yet been successful. Markers associated with the dysplasia-cancer sequence have been reported, including aneuploidy,12 p53,13 microsatellite instability,14 and the mucin-associated sialyl-Tn antigen,15 but none have yet been recommended in surveillance management. Genome wide associated studies (GWAS) revealed susceptibility loci and pathways that influence the risk of disease16, 17, but alone are insufficient to determine specific causative relationships18, suggesting that other genetic and/or epigenetic alterations contribute to UC pathogenesis19.

MicroRNAs are small non-coding RNAs, 18 to 25 nucleotides in length, which regulate gene expression through binding in 3′-untranslated region of target mRNAs.20 MicroRNAs are essential regulators of inflammatory signaling pathways,21, 22 contributing to the pathogenesis of different human inflammatory diseases,23-25 including IBD26, 27. Furthermore, manipulation in the expression levels of microRNAs could suppress the inflammatory response,24 suggesting their therapeutic potential. Although microRNA signaling pathways have been reported to link chronic inflammation to malignant transformation in breast cancer and liver cancer,24, 28, 29 such evidence for UC-associated cancer is currently missing. We hypothesized that microRNAs may contribute to the development of UC and progression to colon cancer, while manipulation in the expression levels of these microRNAs could have therapeutic potential in patients with premalignant lesions.

Materials and Methods

Study oversight

The study was approved by the institutional review board at each study center. All patients from whom tissue samples were obtained at UCLA provided written informed consent. The patient samples were obtained at the Leiden University Medical Center according to the instructions and guidelines of the LUMC Medical Ethics Committee and in accordance with the Helsinki Declaration.

MicroRNA library screen

NCM460 immortalized epithelial cells were plated in 96-well plates and transfected with a microRNA inhibitor library consisting of 348 microRNA inhibitors and 2 negative control microRNAs (100 nM) (Dharmacon Inc), as previously described27. At 24 hours post-transfection, the cells were treated with IL6 for 24 hours, and the phosphorylation of NF-κB was assessed by Phospho-RelA/NF-κB p65 (S536) Cell-Based ELISA (KCB7226, R&D). MicroRNA inhibitors that affected >50% the phosphorylation of NF-κB were considered positive hits.

Statistical analysis

All experiments were performed in triplicate unless otherwise stated. Statistical analyses were performed with the use of Origin software, version 8.6 and SAS statistical package version 9.4. Student's t test was used to examine the statistical difference in miR-214 expression between control colonic tissues and specimens derived from different intestinal pathologies, between active and inactive UC specimens, between UC specimens categorized based on disease duration and between the differentially treated cells and mice groups. The Kruskall Wallis test was used for age comparison and posthoc Wilcoxon two sample test with Bonferroni significance level adjustment for multiple testing were used for age comparisons, Chi-square test for gender comparisons and Fisher's Exact test for tumor stage comparison. The correlation significance was determined by means of Spearman and Pearson correlation analyses. A P value of 0.05 or less was considered to indicate statistical significance.

Additional Materials and Methods are included in the supplementary material.

Results

High throughput inhibition of the human microRNAome in human colonocytes reveals novel regulators of the NF-κB activity

Ulcerative Colitis (UC) development is characterized by activation of inflammatory pathways, including NF-κB signaling. To identify microRNAs that regulate the NF-κB activity in human colonocytes, we performed a microRNA functional screen by targeting the human microRNAome in IL6-treated NCM-460 colonic epithelial cells (Figure 1A). In the primary screen, we found 9 microRNA inhibitors to regulate NF-κB phosphorylation levels by >50% (P<0.05). Specifically, we found two microRNA inhibitors (miR-26b and miR-199a) that induced significantly NF-κB phosphorylation levels, while seven inhibitors (miR-7, miR-146a, miR-373, miR-372, miR-181b, miR-21 and miR-214) suppressed NF-κB phosphorylation levels (Figure 1B). Among these inhibitors, previous studies have shown that miR-21, miR-181b and miR-146a are essential regulators of the inflammatory response, validating our functional screen assay22, 30. Interestingly, our screen revealed that the inhibitor against miR-214, was the most efficient (>90%) suppressor of NF-κB phosphorylation, while the inhibitor against miR-199a was the most efficient (>90%) activator of NF-κB phosphorylation in human colonocytes.

Fig. 1. High-content inhibitor screening identifies microRNAs that control NF-κB in ulcerative colitis.

Fig. 1

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(A) Screening workflow: A library of 348 microRNA inhibitors was transfected in colonic epithelial cells in triplicates and the activation of NF-κB was measured after treatment with IL-6. (B) Screen data plotted as absolute levels (x-axis) of phosphorylated NF-κB (on S536), compared to scrambled sequence controls (no effect, value=1.34) and P value from Student's t test (y-axis). Box shows microRNA inhibitors that affect NF-κB phosphorylation by more than 50%. (C and D) MiR-214 is specifically overexpressed in colonic tissues from patients with UC. (C) MicroRNAs involved in the activation of NF-κB were quantified in colonic specimens by quantitative real time polymerase chain reaction (qPCR). (D) MiR-214 is overexpressed in colonic tissues from patients with UC from three different patient cohorts, as assessed by qPCR. IBS, irritable bowel syndrome; CD, Crohn's disease. Data are represented as mean ± SE. ***P<0.001, **P<0.01 in comparison to control; #P<0.001, in comparison to UC, Student's t test. (E) MiR-214 is upregulated in the colonic epithelium of human active UC samples. In situ hybridization indicates the expression of miR-214 (blue) in the colonic epithelium of UC and control subjects. Nuclear Red was used as counterstain. Scale bars, 50 μm. (F) MiR-214 expression correlates with UC activity. Data are represented as mean ± SE. ***P<0.001, in comparison to UC patients in remission, Student's t test

MiR-214 is specifically overexpressed in UC colonic tissues

According to our microRNA inhibitor screen data, in human colonocytes seven microRNA inhibitors have the ability to significantly suppress the inflammatory response. Next, our aim was to evaluate the clinical relevance of these in vitro findings and assess the expression levels of these microRNAs in human colonic tissues derived from UC and CD patients and control subjects (Figure 1C). Realtime PCR analysis revealed that miR-21 and miR-146a expression is increased in both UC and CD colonic tissues relative to controls, consistent with previous studies31. On the other hand miR-7, miR-181b, miR-372 and miR-373 were not significantly deregulated in UC or CD relative to control subjects. Interestingly, we found that miR-214 is highly (>8-fold) up-regulated specifically in UC colonic tissues relative to control subjects and CD patients tissues. To further validate these findings in a large number of patient samples, we examined miR-214 levels in UC (n=120) and control (n=107) colonic tissues from 3 different cohorts (Tables S1 and S2). Consistent with our initial findings, miR-214 levels were found to be significantly increased in UC, but not irritable bowel syndrome (IBS) (n=22) or CD (n=60) (Figure 1D). In situ hybridization in human colonic tissues identified epithelial cells as the origin of miR-214 overexpression (Figure 1E and S1).

MiR-214 expression correlates with UC disease activity

To evaluate the clinical relevance of our findings, we examined miR-214 expression in correlation with different clinicopathological parameters. MiR-214 levels were significantly higher in UC patients with active disease relative to patients in remission (Figure 1F), but did not differ in relation to UC patient's gender or anatomic site, neither to gender/disease location in CD patients (Figure S2). Taken together, these results demonstrate that MIR214 is an epithelial expressed gene that regulates NF-κB activity and is deregulated specifically in UC in correlation with disease activity.

Integration of computational and molecular analyses for identification of miR-214 downstream targets in colitis

To establish the molecular link between miR-214 and UC development, we aimed to identify downstream gene targets that could mediate NF-κB activation in human colonocytes. We have developed an innovative strategy, consisting of five different steps in order to identify the key effectors of miR-214 activity in UC (Figure 2A). Specifically, during the first step we employed 4 different microRNA target prediction softwares and identified 280 common miR-214 gene targets, based on their sequence complementarity. The second step was to analyze the expression of the genes regulated by miR-214 overexpression in human colonocytes, by performing high throughput expression analysis. This analysis revealed a signature of 71 genes (Table S3) that were differentially expressed (expression <50%, P<0.01) in miR-214-treated NCM460 colonocytes. The next steps included evaluation of these 71 genes for their correlation with the NF-κB signaling pathway and direct interaction with miR-214 employing 3′ untranslated region (3′UTR) luciferase assays. Finally, these direct interactions between miR-214 and its target genes were validated in a second colonic epithelial cell line.

Fig. 2. MiR-214 targets PDLIM2 and PTEN and activates NF-κB in the colonic epithelium.

Fig. 2

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(A) Strategy for the identification and validation of miR-214 targets regulating NF-κB. MiR-214 targets predicted by 4 different softwares (n=280). Gene expression analysis validated 71 miR-214 direct targets inhibited by more than 50% (** P<0.01). Targets were sorted based on relation to NF-κB pathway and alignment of the 3′UTR of mRNAs and miR-214 sequence. (B) MiR-214 targets the 3′UTR of PDLIM2 and PTEN mRNAs. Luciferase assays performed in a second colonic epithelial cell line, NCM460, showed significant inhibition of the 3′UTR reporter activities by miR-214. Data are shown as the mean ± SE. ***P<0.001, **P<0.01, in comparison to untreated and control (scramble) miR-treated cells, Student's t test. (C) MiR-214 regulates the mRNA levels of PDLIM2 and PTEN in colonic epithelial cells. Effect of miR-214 on the expression of PDLIM2 and PTEN in NCM460 cells, as assessed by qPCR. Data are shown as the mean ± SE. **P<0.01, in comparison to untreated and control (scramble) miR-treated cells, Student's t test. (D) MiR-214 regulates the protein levels of PDLIM2 and PTEN in colonic epithelial cells. Western blot analysis of PDLIM2 and PTEN protein levels in NCM356 from three independent replicates. α-Tubulin was used as loading control. (E) MiR-214 regulates the phosphorylation of NF-κB on S536. Data are shown as the mean ± SE of phosphorylated NF-κB (on S536) levels, as assessed by ELISA. **P<0.01, in comparison to untreated and control (scramble) miR-treated cells, Student's t test. (F) PDLIM2 (P<0.01) and PTEN (P<0.05, Student's t test) expression is downregulated in colonic tissues from UC patients.

PDLIM2 and PTEN are direct downstream effectors of miR-214 in UC

The comprehensive analysis described above, revealed two genes that fulfill all the criteria as direct targets of miR-214 in UC. Specifically, PDLIM2 and PTEN were found differentially expressed in miR-214 overexpressing colonocytes, possessing target sequences for miR-214 in their 3′UTRs (Figure 2A). PDLIM2 is a nuclear ubiquitin E3 ligase and a known inhibitor of NF-κB activity 32, and PTEN is a suppressor of the AKT signaling pathway 33 shown to intervene with NF-κB activation34, 35 and result in increased severity of colitis36. To validate the direct interactions at the molecular level between miR-214 and PTEN or PDLIM2, we performed luciferase assays. Specifically, miR-214 was overexpressed in cells together with a luciferase vector harboring the 3′UTR of PTEN or PDLIM2 mRNAs. This analysis revealed that miR-214 overexpression inhibited both PTEN and PDLIM2 3′UTR luciferase activities (Figure 2B). Furthermore, miR-214 overexpression suppressed PTEN and PDLIM2 mRNA (Figure 2C) and protein levels (Figure 2D). In addition, overexpression of miR-214 induced NF-κB (s536) phosphorylation (Figure 2E) and subsequently the expression of IL6 (Figure S3), indicating that PTEN and PDLIM2 are direct downstream effectors of miR-214 involved in the regulation of the NF-κB inflammatory response. Finally, in reflection of the human relevance of these findings, PTEN and PDLIM2 mRNA levels were decreased in UC colonic tissues relative to controls (Figure 2F).

STAT3 transcription factor regulates miR-214 expression in human colonocytes

Given that miR-214 is significantly upregulated in UC, we were interested to identify the molecular mechanisms that regulate miR-214 expression. We performed bioinformatics analysis using the Lever/PhylCRM algorithm which predicts highly conserved binding sites in promoter areas of genes37. This algorithm predicted that the strongest conserved binding site in the MIR214 promoter area is for the transcription factor STAT3 (Figure 3A). To validate at the molecular level this prediction we performed a dual experimental approach. First, we performed chromatin immunoprecipitation (ChIP) analysis by using an antibody against STAT3 and performed qPCR upon stimulation with IL6, using primers specific for the MIR214 promoter area. This experiment showed that STAT3 binds directly, in an IL6 dose response rate, on the MIR214 promoter. In agreement, qPCR analysis revealed the IL6 dose-dependent induction of miR-214 expression (Figure 3A). In the second approach, miR-214 promoter area was cloned in a reporter vector upstream of the luciferase gene. We found that IL6 induced MIR214 promoter activity in a dose-dependent manner, an effect abolished by mutation of the STAT3 binding site (Figure 3B).

Fig. 3. MiR-214 activates an inflammatory feedback loop circuit in the colonic epithelium.

Fig. 3

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(A and B) Inflammatory signals transcriptionally activate miR-214 through STAT3. (A) The Lever and PhylCRM algorithms revealed the presence of STAT3 binding site on the promoter of MIR214 gene (upper panel). IL6 induces dose-dependently the binding of STAT3 on the promoter of MIR214 gene, as assessed by ChIP and qPCR analysis (left panel), and the expression of miR-214, as assessed by qPCR (right panel). Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells, Student's t test. (B) STAT3 binding on the promoter is required for the transcriptional activation of MIR214 gene. IL6 induces the luciferase activity of the wild-type (WT) MIR214 gene promoter, while mutagenesis of the STAT3 binding site abolishes promoter activation by IL6. WT, wild type and M, mutated STAT3 binding site. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells; #P<0.001, in comparison to the IL6-induced WT promoter activity. (C) IL6 suppresses PDLIM2 and PTEN expression in colonocytes through miR-214. Effect of miR-214 inhibition on IL6-induced PDLIM2 and PTEN suppression, as assessed by qPCR. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells, @P<0.01 in comparison to negative control (NC) miR-treated cells, Student's t test. (D) Schematic representation of the proposed model. (E and F) Activation and inhibition of the inflammatory feedback loop circuit. (E) miR-214 overexpression activates STAT3 while silencing of miR-214 abrogates the IL6-induced activation of STAT3. Western blot analysis of PDLIM2 and PTEN protein levels and the phosphorylation levels of STAT3 and NF-κB in NCM356 cells. α-Tubulin was used as loading control. (F) Pharmacological inhibition of Akt or NF-κB reverses the IL6-induced suppression of PDLIM2 and PTEN, as assessed by qPCR. Data are shown as the mean ± SE. ***P<0.001, **P<0.01, *P<0.05 in comparison to IL6-treated cells, Student's t test.

MiR-214 is a part of a feedback loop circuit mediated by PDLIM2 and PTEN genes

The role of miR-214 as inflammatory mediator through the regulation of PDLIM2 and PTEN was studied in NCM356 human colonocytes. IL6 dose-dependently suppressed PDLIM2 and PTEN mRNA levels an effect reversed by the inhibition of miR-214 (Figure 3C). In accord, Akt phosphorylation was increased upon IL6 treatment, and reduced by miR-214 inhibition (Figure S4). These findings suggest that IL6 induces miR-214 through STAT3-mediated transcriptional activation, creating a positive feedback loop circuit (Figure 3D). Specifically, miR-214 overexpression, suppresses directly the expression levels of PTEN and PDLIM2 genes. The suppression of PDLIM2 results in direct activation of NF-κB, while the suppression of PTEN, results in activation of the AKT kinase, contributing to the phosphorylation of NF-κB. In turn, the NF-κB transcription factor induces IL6 expression and thus activates STAT3 transcription factor, which binds directly on the MIR214 gene promoter area, re-enforcing its up-regulation. To assess the activity of the feedback loop circuit we either overexpressed the microRNA or treated cells with IL6 in combination with miR-214 silencing. Western blot analysis, of cell lysates collected 96h later, revealed the sustained suppression of PTEN and PDLIM2 and increased phosphorylation of NF-κB and STAT3 upon miR-214 overexpression or treatment with IL6. Importantly, miR-214 targeting reversed the effects of IL6, in support of the dependence of circuit activation on miR-214 (Figure 3E). In the same line, pharmacological inhibition of Akt or NF-κB resulted in partial or complete reversal, respectively, of the IL6-induced effects on PTEN and PDLIM2 expression (Figure 3F).

Effects of a miR-214 chemical inhibitor on UC ex vivo and in vivo

To evaluate the therapeutic potential of miR-214 inhibition in UC through regulation of the feedback loop circuit, we used a chemically stable inhibitor, highly efficient in suppressing miR-214 in human colonocytes in vitro (Figure 4A). We studied the effects of the miR-214 chemical inhibitor ex vivo on freshly isolated colonic biopsies from UC patients with active disease (Figure 4B). The inhibitor efficiently inhibited miR-214 in the colonic explants (Figure 4C), and suppressed the inflammatory response by increasing the expression of both PDLIM2 and PTEN in colonic explants from 3 UC patients with active disease (Figure 4D). In addition to the ex vivo findings, we examined the effects of the miR-214 inhibitor on experimental colitis in mice. Mice treated with DSS and the miR-214 inhibitor and colitis disease activity was evaluated by using different parameters (Figure 4E). Importantly, we found that the miR-214 inhibitor reduced disease activity in DSS-induced experimental colitis (Figure 4F). Taken together, these in vitro, in vivo and ex vivo data suggest the therapeutic potential of the miR-214 inhibitor in UC patients with active disease.

Fig. 4. A chemical inhibitor reverses the effects of miR-214 on colonic tissues from UC patients and ameliorates disease activity in a mouse colitis model.

Fig. 4

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(A) A chemical inhibitor effectively suppresses miR-214 expression in colonocytes in vitro. The effectiveness of a miR-214-specific chemical inhibitor was tested in vitro on NCM356 colonocytes. Cells treated with the miR-214 chemical inhibitor (for 30 min) were analyzed 24 hours later for miR-214 levels, as assessed by qPCR. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated and negative control treated cells, Student's t test. (B) Fresh colonic biopsies isolated at colonoscopy were divided in two and treated with the chemical inhibitor against miR-214 or negative control (NC). (C) A chemical inhibitor effectively suppresses miR-214 expression in colonic biopsies ex vivo. Following treatments, RNAs were extracted from biopsies and analyzed for miR-214 levels by qPCR. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated and negative control treated biopsies, Student's t test. (D) A chemical inhibitor reverses miR-214 effects in colonic biopsies ex vivo. Biopsies were analyzed for PDLIM2 and PTEN levels by qPCR. (E) The therapeutic protocol applied on the chronic DSS mouse model and the he criteria used for assessment of the disease activity score. miR-214 inhibitor or the negative control, were intracolonically administered in DSS-treated mice after day 8 for four cycles (every 2 days). On day 20, the mice were sacrificed, and colon length was measured. (F) A chemical inhibitor of miR-214 ameliorates disease activity in the chronic DSS mouse colitis model. Data are shown as the mean ± SE. **P<0.01 in comparison to untreated and NC-treated DSS mice, 4 mice per group, Student's t test.

MiR-214 is overexpressed in long-lasting colitis and colitis-associated colon cancer

Although the association between UC and colorectal cancer has been documented as early as 192538, its molecular biology remains to be explored. We questioned the role of miR-214 during the transition from UC to cancer. Interestingly, we found that miR-214 is up-regulated in mice with chronic but not acute inflammation induced by DSS (Figure 5A). In the same line, colonic tissues from patients with longstanding (>10 years) UC have increased miR-214 levels compared to those with <10 years (Figure 5B), suggesting that miR-214 may provide a link between UC and colorectal cancer development. Notably, miR-214 levels do not change significantly in colonic tissues of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated mice as well as in polyps formed in the adenomatous polyposis coli mutant (APCmin) mouse model (Figure S5). Therefore, we compared miR-214 levels by qPCR and in situ hybridization in tumors from CAC and sporadic colon cancer (CRC) patients. This analysis showed a dramatic over-expression of miR-214 in CAC, but no difference between CRC and controls (Figure 5C, D and S6). Overall, these findings suggest that miR-214 is increased during UC development and amplified during progression to colorectal cancer.

Fig. 5. MiR-214 expression correlates with the duration of ulcerative colitis and the development of colitis-associated cancer.

Fig. 5

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(A) Chronic colonic inflammation regulates miR-214 expression in mice. Data are shown as the mean ± SE ***P<0.001 in comparison to control (DSS-untreated) mice. (B) Chronic colonic inflammation regulates miR-214 expression in UC patients. Data are shown as the mean ± SE. **P<0.01, ***P<0.001 in comparison to control (No UC) tissues. (C) miR-214 is hyperexpressed in colitis-associated colorectal cancer (CAC), but not sporadic colorectal cancer (CRC), as demonstrated by qPCR. Data are shown as the mean ± SE. ***P<0.001, in comparison to control tissues; #P<0.001, in comparison to CAC, Student's t test. (D) In situ hybridization indicates the overexpression of miR-214 (blue) in tumors from CAC compared to CRC patients. Nuclear Red was used as counterstain. Scale bars, 50 μm.

MiR-214 as a novel oncogene in colon cancer

To examine the significance of miR-214 in colorectal oncogenesis we performed gain- and loss-of function studies. Overexpression of miR-214 induced the colony formation ability (Figure 6A, S7A) and invasiveness (Figure 6B, S7B) of human colon cancer cells, suggesting its oncogenic role. On the other hand, silencing of miR-214 suppressed the IL6-induced tumorigenic and invasive phenotype, pointing to the dependence of IL6 tumorigenic effects on miR-214. Next, we investigated whether miR-214 acts through regulation of the feedback loop circuit. We found that IL6 up-regulated miR-214 in HCT-116, HT-29, LoVo and DLD-1 colon cancer cells (Figure 6C), mediated by the direct binding of STAT3 on MIR214 promoter area (Figure 6C, D). Furthermore, IL6-induced miR-214 expression resulted in PDLIM2 and PTEN suppression (Figure S8), Akt phosphorylation (Figure S9) and NF-κB activation (Figure S10), indicating that the miR-214 feedback loop circuit is hyper-activated during oncogenesis. MiR-214 overexpression suppressed PTEN and PDLIM2 mRNA levels (Figure S11) and in support of the human relevance, PDLIM2 and PTEN levels were significantly decreased in tumors from CAC patients relative to controls (Figure S12). In contrast, their levels were not significantly decreased in tumors from CRC patients, supporting the specificity of miR-214 circuit in CAC (Figure 6E). Given that miR-214 increases cancer cell aggressiveness, we examined miR-214 correlation to colon cancer progression. Examination of human tumors indicated that miR-214 is specifically up-regulated during CAC disease progression (Figure 6F), providing additional evidence on the specificity of miR-214 circuit in UC and CAC.

Fig. 6. Tumorigenic potential of the miR-214 circuit in human colonocytes.

Fig. 6

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(A) and (B) IL-6 through miR-214 regulates the tumorigenic properties of colon cancer cells. (A) MiR-214 overexpression (pre-miR-214) enhances colon cancer cell colony formation in soft agar, whereas miR-214 inhibition (as-miR-214) reverses the effects of IL6. Scale bars, 500 μm. (B) MiR-214 overexpression (pre-miR-214) enhances colon cancer cell invasion in matrigel, whereas miR-214 inhibition (as-miR-214) reverses the effects of IL6. Scale bars, 100 μm. Data are shown as the mean ± SE. **P<0.01, ***P<0.001 in comparison to untreated and control (pre-miR-C)-treated cells; #P<0.001 in comparison to as-miR-negative control (NC)-treated cells; Student's t test. (C) IL6 induces dose-dependently the expression of miR-214 and the binding of STAT3 on the promoter of MIR214 gene, in colon cancer cells. Effects of IL6 on the expression of miR-214, as assessed by qPCR and STAT3 binding as assessed by ChIP and qPCR analysis. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells, Student's t test. (D) STAT3 binding on the promoter is required for the transcriptional activation of MIR214 gene in colon cancer cells. IL6 induces the luciferase activity of the wild-type (WT) MIR214 gene promoter, while mutagenesis of the STAT3 binding site abolishes promoter activation by IL6. WT, wild type and M, mutated STAT3 binding site. Data are shown as the mean ± SE. ***P<0.001, in comparison to untreated cells; #P<0.001, @P<0.01, in comparison to the IL6-induced WT promoter activity, Student's t test. (E) PDLIM2 and PTEN expression is significantly suppressed in CAC but not CRC, as assessed by qPCR. Data are shown as the mean ± SE. **P<0.01, ***P<0.001 in comparison to control colonic tissues, Student's t test. (F) MiR-214 levels positively correlate with tumor staging in CAC. Data are shown as the mean ± SE. *P<0.05, in comparison to early CAC stages (I and II), Student's t test.

MiR-214 chemical inhibitor suppresses AOM-DSS-induced colon carcinogenesis

According to our data the miR-214-positive feedback loop circuit links chronic inflammation to colon carcinogenesis. Therefore, targeting miR-214 may suppress the development of CAC. We applied a therapeutic protocol consisting of intracolonic administration of a chemical miR-214 inhibitor or microRNA negative control for 4 cycles, weekly (Figure 7A, S13) on the AOM-DSS mouse model. The miR-214 inhibitor reduced significantly the number (Figure 7B) and size (Figure 7C) of tumors. Mechanistically, miR-214 inhibitor suppressed tumor growth through the induction of apoptosis and activation of caspase-3 (Figure 7D). In addition, immunohistochemical analysis revealed that miR-214 inhibition reduced NF-κB and Akt phosphorylation levels, indicating efficient suppression of the miR-214-molecular circuit, and decreased the proliferation rate of colon cancer cells (Figure 7E).

Fig. 7. A chemical inhibitor of miR-214 effectively suppresses colitis-associated colorectal cancer in vivo.

Fig. 7

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(A) Protocol for anti-miR-214 therapy in the mouse AOM-DSS colitis-associated colorectal cancer model. (B to D) Evaluation of the therapeutic potential of intracolonic administration of miR-214 inhibitor on the (B) number and (C) size of tumors, and (D) the levels of cleaved caspase 3 in the tumors formed in the AOM-DSS mouse model of colitis-associated colorectal cancer. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated and control (miR-NC inhibitor)-administered mice, 10 mice per group, Student's t test. (E) Haematoxylin/eosin, pNF-κB (S536), pAkt (S473), and Ki67 staining in sections of colonic tissues from mice treated with the chemical miR-214 inhibitor or negative control (NC). Scale bars, 200 μm (for H&E staining) and 50 μm (for immunohistochemistry). (F) Schematic representation of the activity and role of miR-214 molecular circuit in UC and CAC. In healthy non-inflamed colonic epithelial cells PDLIM2 and PTEN expression suppresses the activation of Akt and NF-κB. During the development of UC, mir-214 targets PDLIM2 and PTEN to activate Akt and NF-κB. In turn, NF-κB regulates IL6 expression and thus STAT3 activity. STAT3-mediated transcriptional activation of miR-214 creates a positive feedback loop circuit which is attenuated when disease is in inactive state. In longstanding UC, overexpression of miR-214, and hyperactivation of this inflammatory circuit promotes the development of colorectal cancer.

Discussion

MiR-214 expression correlates with UC activity and disease duration and provides the molecular rationale for the current practice guidelines recommending surveillance colonoscopy 8 to 10 years after diagnosis of UC39. Although the association between IBD and cancer, linking colonic inflammation to colorectal cancer risk, has been documented as early as 1925,38 its molecular biology remains unknown. We propose that a microRNA, miR-214, is the central regulator, based on its unique expression pattern in ulcerative colitis-associated colon cancer patients. Our findings suggest that miR-214 could serve as a biomarker identifying patients at risk for this malignant transformation. We also show that miR-214 expression correlates well with disease activity and disease duration, which offers the molecular rationale for the current practice guidelines recommending surveillance colonoscopy for UC patients 8 to 10 years after diagnosis.39

Clinical and epidemiological studies have revealed a strong association between chronic inflammation and development of cancer through activation of inflammatory molecules.40, 41 IL6 is secreted by epithelial and immune cells contributing to the transformation of normal into malignant cells24, 28, 42. Although inflammatory responses have been found to be activated in both sporadic and colitis-associated colon cancers, it is not clear whether these inflammatory signals regulate the same signaling pathways. Our data reveal miR-214 as an inflammatory effector molecule that is deregulated in colitis-associated colon cancer patients. The STAT3 transcription factor is able to bind to the promoter area of miR-214 and the presence of IL6, leads to IL6-STAT3 dependent miR-214 expression and thus the regulation of both the PDLIM2-NF-κB and the PTEN-Akt pathways. Also, miR-214 up-regulation induces NF-κB-IL6 pathway suggesting the presence of a miR-214 feedback loop circuit. Gain- and loss-of function assays confirmed the significance of miR-214 in colorectal oncogenesis. Given that IL6-STAT3 signaling is activated while miR-214 expression in other GI diseases, such as CD and CRC, we propose that the responsiveness of MIR214 gene to inflammatory signals depends on epigenetic cues deregulated upon sustained chronic colonic inflammation. Although, we have identified feedback loop inflammatory circuits in different human diseases 24, 28, this is the first demonstration of a feedback loop mechanism that is involved not only in two human diseases (ulcerative colitis and colon cancer), but importantly, its acts as a rheostat, being hyper-activated and contributing to the progression from colitis to colon cancer (Figure 7F).

Therapeutic applications are conceivable as demonstrated in our experiments by using the novel miR-214 chemical inhibitor. Studies have shown these microRNA mimics or microRNA inhibitors have a therapeutic potential in different types of cancer and auto-immune diseases. With the recent demonstration that inhibition of miR-122 reduces viral load in HCV-infected chimpanzees,43 miR-122 inhibitors are already in Phase II trials. We have recently demonstrated that intravenous administration of a microRNA mimic could have therapeutic potential in liver cancer patients through suppression of the inflammatory response.24 Here, we show that intra-colonic administration of a chemically modified antisense-miR-214 suppresses effectively the development of tumor growth in an animal model of colitis-associated colon cancer, suggesting that a similar approach could be considered for colitis-associated dysplasia and cancer.

Supplementary Material

Figure S1. miR-214 is upregulated in the colonic epithelium of human active UC samples. Quantification of miR-214 staining using in situ hybridization. Scoring was performed independently by two investigators, blind to the sample IDs. N=4 per group.

Figure S2. miR-214 expression in patients with ulcerative colitis and Crohn's disease. miR-214 expression, as assessed by qPCR, in UC patients does not correlate to the anatomical site of the colon (A) or gender (B), and does not differ in CD patients based on (C) disease location or (D) gender. Data are shown as the mean ± SE.

Figure S3. miR-214 regulates the expression of IL6 in colonic epithelial cells. Effect of miR-214 on the expression of IL6 in NCM460 cells, as assessed by qPCR. Data are shown as the mean ± SE. ***P< 0.001 in comparison to untreated and control (scramble) miR-treated cells, Student's t test.

Figure S4. IL6 induces Akt phosphorylation in colonocytes through miR-214. Effect of miR-214 inhibition on IL6-induced Akt phosphorylation (S473), as assessed by ELISA assay. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells, #P<0.001 in comparison to NC miR-treated cells, Student's t test.

Figure S5. miR-214 levels are not deregulated in other inflammation-associated mouse models. (A) TNBS-induced colonic inflammation does not deregulate miR-214 expression. Data are shown as the mean ± SE, in comparison to control (TNBS-untreated) mice, 4 mice per group. (B) Polyp formation in the APC min mouse model does not involve changes in the expression of miR-214. Data are shown as the mean ± SE, in comparison to control (no polyps) tissues, 5 tissues per group.

Figure S6. miR-214 is significantly upregulated in colitis-associated colorectal cancer compared to sporadic colorectal cancer. Quantification of miR-214 staining using in situ hybridization. Scoring was performed independently by two investigators, blind to the sample IDs. N=4 for CRC and 9 for CAC.

Figure S7. miR-214 induces the tumorigenic potential of colon cancer cells and mediates the tumorigenic effects of IL6. miR-214 overexpression enhances the tumorigenic potential, while miR-214 inhibition reverses the tumorigenic effects of IL6, on colon cancer cells. Effects of miR-214 overexpression and miR-214 inhibition on IL6-induced (A) colony formation in soft agar, and (B) invasion of SW480 cells. Data are shown as the mean ± SE. **P <0.01, ***P<0.001 in comparison to untreated cells and control (miR-scramble)-treated cells and #P<0.001 in comparison to anti-miR-negative control (NC)-treated cells, Student's t test.

Figure S8. IL6 suppresses the expression of PDLIM2 and PTEN in colon cancer cells through miR-214. Effects of IL6 and inhibition of miR-214 on the expression of (A) PDLIM2 and (B) PTEN mRNAs, as assessed by qPCR, in HCT116 and HT29 cells. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells. @P<0.01 in comparison to the anti-miR-negative control (NC)-treated cells, Student's t test.

Figure S9. IL6 induces the phosphorylation of Akt in colon cancer cells through miR-214. Effect of miR-214 inhibition (as-miR-214) on IL6-induced Akt phosphorylation (S473), as assessed by ELISA. Data are shown as the mean ± SE. **P<0.01, ***P<0.001 in comparison to untreated cells, #P<0.001 in comparison to control (as-miR-NC)-treated cells, Student's t test.

Figure S10. MiR-214 regulates the phosphorylation of NF-κB in colon cancer cells. Effects of miR-214 on the levels of pNF-κB (S536) in HCT116 cells, as assessed by ELISA. Data are shown as the mean ± SE. ***P<0.001, in comparison to untreated and control (pre-miR-C)-treated cells, Student's t test.

Figure S11. MiR-214 regulates the expression of PDLIM2 and PTEN in colon cancer cells. Effects of miR-214 on the levels of PDLIM2 and PTEN mRNAs in HCT116 cells, as assessed by qPCR. ***P<0.001, in comparison to untreated and control (pre-miR-C)-treated cells, Student's t test.

Figure S12. PDLIM2 and PTEN expression is downregulated in colon tumors from CAC patients. PDLIM2 and PTEN mRNA levels in CAC and control colonic tissues, was assessed by qPCR.

Figure S13. The therapeutic protocol applied on AOM-DSS mouse model. To address the therapeutic potential of miR-214 inhibition we used a mouse model of colitis-associated colorectal cancer. In this model colorectal cancer is induced by administration of azoxymethane (AOM) followed by repeated oral administration of dextran sulfate sodium (DSS). Chemical microRNA negative control (miR-NC) or miR-214 inhibitor were intracolonically administered in AOM-DSS-treated mice on a weekly basis for four cycles. On day 91, the mice were sacrificed, and tumor burden was assessed.

Table S1. Cohorts of patients analyzed for miR-214 expression

Table S2. Characteristics of patients analyzed for miR-214 expression

Table S3. Expression of miR-214 targets vs Control (miR-scramble) in colonic epithelial cells

Acknowledgments

Grant Support: This work was in part supported by the National Center for Advancing Translational Sciences UCLA CTSI Grant UL1TR000124 and the Broad Medical Research Program at CCFA (CP1), Cancer Research Institute Investigator Award (DI), The Leona M. and Harry B. Helmsley Charitable Trust (DI) and NIH National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK60729 (CP3).

Footnotes

Author contributions: Study concept (DI); study design (CP1, DI); data acquisition (CP1, TP, MH, GK, AO, CV, MK, EB); clinical data and sample collection (DH, WD, OS, JC, LC, PA, PT, CP3, HV); statistical analysis (CP1, MH, WD); data interpretation and manuscript drafting (CP1, DH, CP3, DI); critical revision of the manuscript (DI).

Conflict of Interest: None to declare for all the authors.

Author names in bold designate shared co-first authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. miR-214 is upregulated in the colonic epithelium of human active UC samples. Quantification of miR-214 staining using in situ hybridization. Scoring was performed independently by two investigators, blind to the sample IDs. N=4 per group.

Figure S2. miR-214 expression in patients with ulcerative colitis and Crohn's disease. miR-214 expression, as assessed by qPCR, in UC patients does not correlate to the anatomical site of the colon (A) or gender (B), and does not differ in CD patients based on (C) disease location or (D) gender. Data are shown as the mean ± SE.

Figure S3. miR-214 regulates the expression of IL6 in colonic epithelial cells. Effect of miR-214 on the expression of IL6 in NCM460 cells, as assessed by qPCR. Data are shown as the mean ± SE. ***P< 0.001 in comparison to untreated and control (scramble) miR-treated cells, Student's t test.

Figure S4. IL6 induces Akt phosphorylation in colonocytes through miR-214. Effect of miR-214 inhibition on IL6-induced Akt phosphorylation (S473), as assessed by ELISA assay. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells, #P<0.001 in comparison to NC miR-treated cells, Student's t test.

Figure S5. miR-214 levels are not deregulated in other inflammation-associated mouse models. (A) TNBS-induced colonic inflammation does not deregulate miR-214 expression. Data are shown as the mean ± SE, in comparison to control (TNBS-untreated) mice, 4 mice per group. (B) Polyp formation in the APC min mouse model does not involve changes in the expression of miR-214. Data are shown as the mean ± SE, in comparison to control (no polyps) tissues, 5 tissues per group.

Figure S6. miR-214 is significantly upregulated in colitis-associated colorectal cancer compared to sporadic colorectal cancer. Quantification of miR-214 staining using in situ hybridization. Scoring was performed independently by two investigators, blind to the sample IDs. N=4 for CRC and 9 for CAC.

Figure S7. miR-214 induces the tumorigenic potential of colon cancer cells and mediates the tumorigenic effects of IL6. miR-214 overexpression enhances the tumorigenic potential, while miR-214 inhibition reverses the tumorigenic effects of IL6, on colon cancer cells. Effects of miR-214 overexpression and miR-214 inhibition on IL6-induced (A) colony formation in soft agar, and (B) invasion of SW480 cells. Data are shown as the mean ± SE. **P <0.01, ***P<0.001 in comparison to untreated cells and control (miR-scramble)-treated cells and #P<0.001 in comparison to anti-miR-negative control (NC)-treated cells, Student's t test.

Figure S8. IL6 suppresses the expression of PDLIM2 and PTEN in colon cancer cells through miR-214. Effects of IL6 and inhibition of miR-214 on the expression of (A) PDLIM2 and (B) PTEN mRNAs, as assessed by qPCR, in HCT116 and HT29 cells. Data are shown as the mean ± SE. ***P<0.001 in comparison to untreated cells. @P<0.01 in comparison to the anti-miR-negative control (NC)-treated cells, Student's t test.

Figure S9. IL6 induces the phosphorylation of Akt in colon cancer cells through miR-214. Effect of miR-214 inhibition (as-miR-214) on IL6-induced Akt phosphorylation (S473), as assessed by ELISA. Data are shown as the mean ± SE. **P<0.01, ***P<0.001 in comparison to untreated cells, #P<0.001 in comparison to control (as-miR-NC)-treated cells, Student's t test.

Figure S10. MiR-214 regulates the phosphorylation of NF-κB in colon cancer cells. Effects of miR-214 on the levels of pNF-κB (S536) in HCT116 cells, as assessed by ELISA. Data are shown as the mean ± SE. ***P<0.001, in comparison to untreated and control (pre-miR-C)-treated cells, Student's t test.

Figure S11. MiR-214 regulates the expression of PDLIM2 and PTEN in colon cancer cells. Effects of miR-214 on the levels of PDLIM2 and PTEN mRNAs in HCT116 cells, as assessed by qPCR. ***P<0.001, in comparison to untreated and control (pre-miR-C)-treated cells, Student's t test.

Figure S12. PDLIM2 and PTEN expression is downregulated in colon tumors from CAC patients. PDLIM2 and PTEN mRNA levels in CAC and control colonic tissues, was assessed by qPCR.

Figure S13. The therapeutic protocol applied on AOM-DSS mouse model. To address the therapeutic potential of miR-214 inhibition we used a mouse model of colitis-associated colorectal cancer. In this model colorectal cancer is induced by administration of azoxymethane (AOM) followed by repeated oral administration of dextran sulfate sodium (DSS). Chemical microRNA negative control (miR-NC) or miR-214 inhibitor were intracolonically administered in AOM-DSS-treated mice on a weekly basis for four cycles. On day 91, the mice were sacrificed, and tumor burden was assessed.

Table S1. Cohorts of patients analyzed for miR-214 expression

Table S2. Characteristics of patients analyzed for miR-214 expression

Table S3. Expression of miR-214 targets vs Control (miR-scramble) in colonic epithelial cells

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