Accepted Manuscript MicroRNA214 is Associated with Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-associated Cancer in Mice Christos Polytarchou, Daniel W. Hommes, Tiziana Palumbo, Maria Hatziapostolou, Marina Koutsioumpa, Georgios Koukos, Andrea E. van der Meulen-de Jong, Angelos Oikonomopoulos, Welmoed K. van Deen, Christina Vorvis, Oksana B. Serebrennikova, Eleni Birli, Jennifer Choi, Lin Chang, Peter A. Anton, Philip N. Tsichlis, Charalabos Pothoulakis, Hein W. Verspaget, Dimitrios Iliopoulos PII: DOI: Reference:
S0016-5085(15)00784-2 10.1053/j.gastro.2015.05.057 YGAST 59828
To appear in: Gastroenterology Accepted Date: 30 May 2015 Please cite this article as: Polytarchou C, Hommes DW, Palumbo T, Hatziapostolou M, Koutsioumpa M, Koukos G, van der Meulen-de Jong AE, Oikonomopoulos A, van Deen WK, Vorvis C, Serebrennikova OB, Birli E, Choi J, Chang L, Anton PA, Tsichlis PN, Pothoulakis C, Verspaget HW, Iliopoulos D, MicroRNA214 is Associated with Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-associated Cancer in Mice, Gastroenterology (2015), doi: 10.1053/ j.gastro.2015.05.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
ACCEPTED MANUSCRIPT
MicroRNA214 is Associated with Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-associated Cancer in Mice Short Title: MicroRNA therapeutics for colitis and colon cancer
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Christos Polytarchou1, Daniel W. Hommes2,3*, Tiziana Palumbo1*, Maria Hatziapostolou1*, Marina Koutsioumpa1, Georgios Koukos1, Andrea E. van der Meulen-de Jong2, Angelos Oikonomopoulos1,3, Welmoed K. van Deen2,3, Christina Vorvis1, Oksana B. Serebrennikova4, Eleni Birli1, Jennifer Choi3, Lin Chang5, Peter A. Anton6, Philip N. Tsichlis4, Charalabos
SC
Pothoulakis3, Hein W. Verspaget2, Dimitrios Iliopoulos1# 1
Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of
Medicine, University of California, Los Angeles, Los Angeles CA USA;
2
Department of
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Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands; 3
Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School
of Medicine, University of California, Los Angeles, Los Angeles CA USA; 4Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA; 5Gail and Gerard Oppenheimer Family Center of Neurobiology of Stress, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles CA USA; 6Center for HIV Prevention Research, David Geffen School of
*equal contribution
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Medicine, University of California, Los Angeles, Los Angeles CA USA.
Author contributions: Study concept (DI); study design (CP1, DI); data acquisition (CP1, TP,
EP
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,
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DH, CP3, DI); critical revision of the manuscript (DI).
Conflict of Interest: None to declare for all the authors.
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).
1
ACCEPTED MANUSCRIPT
#
Corresponding author: Dimitrios Iliopoulos, Ph.D., Center for Systems Biomedicine, Division
of Digestive Diseases, David Geffen School of Medicine, University of California at Los Angeles, 650 Charles E. Young Dr., CHS 44-133, Los Angeles, CA 90095-7278. Tel: 310-825-8856; E-
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EP
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SC
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mail: [email protected]
2
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ABSTRACT
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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).
SC
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).
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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.
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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
3
ACCEPTED MANUSCRIPT
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
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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
SC
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
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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 dysplasiacancer 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
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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
18
specific causative relationships , suggesting that other genetic and/or epigenetic alterations contribute to UC pathogenesis19.
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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
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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.
4
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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
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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
SC
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
M AN U
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
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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
EP
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
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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.
5
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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
RI PT
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% (P90%) suppressor of NF-κB phosphorylation, while the inhibitor against miR-199a was the most efficient (>90%) activator of NF-κB phosphorylation in human colonocytes.
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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
EP
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
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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). 6
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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
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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
SC
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
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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
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(expression
S0016-5085(15)00784-2 10.1053/j.gastro.2015.05.057 YGAST 59828
To appear in: Gastroenterology Accepted Date: 30 May 2015 Please cite this article as: Polytarchou C, Hommes DW, Palumbo T, Hatziapostolou M, Koutsioumpa M, Koukos G, van der Meulen-de Jong AE, Oikonomopoulos A, van Deen WK, Vorvis C, Serebrennikova OB, Birli E, Choi J, Chang L, Anton PA, Tsichlis PN, Pothoulakis C, Verspaget HW, Iliopoulos D, MicroRNA214 is Associated with Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-associated Cancer in Mice, Gastroenterology (2015), doi: 10.1053/ j.gastro.2015.05.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
ACCEPTED MANUSCRIPT
MicroRNA214 is Associated with Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-associated Cancer in Mice Short Title: MicroRNA therapeutics for colitis and colon cancer
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Christos Polytarchou1, Daniel W. Hommes2,3*, Tiziana Palumbo1*, Maria Hatziapostolou1*, Marina Koutsioumpa1, Georgios Koukos1, Andrea E. van der Meulen-de Jong2, Angelos Oikonomopoulos1,3, Welmoed K. van Deen2,3, Christina Vorvis1, Oksana B. Serebrennikova4, Eleni Birli1, Jennifer Choi3, Lin Chang5, Peter A. Anton6, Philip N. Tsichlis4, Charalabos
SC
Pothoulakis3, Hein W. Verspaget2, Dimitrios Iliopoulos1# 1
Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of
Medicine, University of California, Los Angeles, Los Angeles CA USA;
2
Department of
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Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands; 3
Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School
of Medicine, University of California, Los Angeles, Los Angeles CA USA; 4Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA; 5Gail and Gerard Oppenheimer Family Center of Neurobiology of Stress, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles CA USA; 6Center for HIV Prevention Research, David Geffen School of
*equal contribution
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Medicine, University of California, Los Angeles, Los Angeles CA USA.
Author contributions: Study concept (DI); study design (CP1, DI); data acquisition (CP1, TP,
EP
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,
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DH, CP3, DI); critical revision of the manuscript (DI).
Conflict of Interest: None to declare for all the authors.
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).
1
ACCEPTED MANUSCRIPT
#
Corresponding author: Dimitrios Iliopoulos, Ph.D., Center for Systems Biomedicine, Division
of Digestive Diseases, David Geffen School of Medicine, University of California at Los Angeles, 650 Charles E. Young Dr., CHS 44-133, Los Angeles, CA 90095-7278. Tel: 310-825-8856; E-
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EP
TE D
M AN U
SC
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mail: [email protected]
2
ACCEPTED MANUSCRIPT
ABSTRACT
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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).
SC
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).
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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.
AC C
EP
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
3
ACCEPTED MANUSCRIPT
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
RI PT
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
SC
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
M AN U
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 dysplasiacancer 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
TE D
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
18
specific causative relationships , suggesting that other genetic and/or epigenetic alterations contribute to UC pathogenesis19.
EP
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
AC C
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.
4
ACCEPTED MANUSCRIPT
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
RI PT
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
SC
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
M AN U
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
TE D
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
EP
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
AC C
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.
5
ACCEPTED MANUSCRIPT
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
RI PT
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% (P90%) suppressor of NF-κB phosphorylation, while the inhibitor against miR-199a was the most efficient (>90%) activator of NF-κB phosphorylation in human colonocytes.
TE D
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
EP
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
AC C
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). 6
ACCEPTED MANUSCRIPT
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
RI PT
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
SC
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
M AN U
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
TE D
(expression
