miRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis

Abstract

Background: Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis.

Methods: Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation.

Results: miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases.

Conclusions: We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.

FIGURE 1

miR-26b expression in human UC and UC-associated carcinomas (UCC) determined by ISH. A, Hematoxylin and eosin (H&E) staining (top panel) and ISH (bottom panel) for miR-26b expression in inactive colitis (A, D), active UC (B, E), and UCC (C, F) at ×40 magnification. G, ISH scores (0–3) of miR-26b from 38 samples of 17 patients, as described in Materials and Methods, presented as the average scores of 2 independent observers (T.T.R. and A.A.). Samples were grouped according to their pathological stage: inactive colitis (n = 6), active UC (n = 16), and UCC (n = 16). H, Individual ISH scores (0–3) of miR-26b, as described in Materials and Methods. Data are presented as average score. Tissue samples were grouped according to their pathological disease status within a single patient. Nine patients had available samples in an “inactive colitis-UCC” sequence, 4 patients had “UC-UCC” sequence, and 1 patient had “inactive-UC” sequence. Four patients having only UCC samples were indicated as a circle. I, Graphical representation of 2-dimensional hierarchical clustering results on miR-26b ISH data and mRNA qPCR profiles of Ki-67 of inactive colitis (n = 6), active UC (n = 16), and UCC (n = 16) samples. RNA expression scores are depicted according to a color scale: red, positive expression; blue, negative expression; and gray, intermediate expression. Dendrogram of samples discriminates between the 4 groups (indicated in purple, green, blue, and orange).

FIGURE 2

miR-26b expression in human UC determined by quantitative PCR. A, Individual relative expression of miR-26b in healthy people with normal uninflamed colon mucosa (n = 15) and patients with active UC (n = 10) in biopsy specimens determined by qRT-PCR. Analyses were performed in triplicates, and the results were normalized using the U6 expression level; P < 0.0001 (unpaired 2-tailed t test). B, Individual relative expression of miR-26b determined by qRT-PCR in healthy people (n = 10) and patients with active UC (n = 6) in blood serum. In 5 healthy samples, miR-26b/U6 was not amplifiable. Analyses were performed in triplicates, and the results were normalized using the U6 expression level, P < 0.0001 (unpaired 2-tailed t test).

FIGURE 3

Expression of miR-26b in AOM/DSS-induced murine model of CRC. A, Upper panel: scheme of 4-stage protocol inducing chronic inflammation and colitis-driven tumors in mice. Lower panel: endoscopic images display the development of tumors in the murine colon after AOM/DSS treatment. B, Individual relative expression of miR-26b in murine serum of control (day 1, n = 15) and AOM + DSS–treated mice (day 67, n = 5) measured by qRT-PCR. Analyses were performed in triplicates, and the results were normalized using the U6 expression level. P = 0.0001 compared with untreated samples (Mann–Whitney U test).

FIGURE 4

MiR-26b targets associated with DAPK. A, Schematic representation of the strategy used to reveal DAPK-interacting proteins connected through miR-26b. B, Venn diagram illustrating candidates overlapping between miR-26b–specific targets and DAPK-interacting partners. C, Relative enrichment of miR-26b targets identified by Ago2-RIP in HCT116 cells. Expression signals of Ago2-bound RNA were normalized to the IgG-bound RNA from the same cell population, and enrichments of miR-26b–transfected versus scrambled miR–transfected cells (negative control [NC]) were calculated using ΔCt method. Ago2-RIP was repeated twice and qRT-PCR performed 3 times in triplicates. Results are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared with scrambled transfection (unpaired 2-tailed t test). D, Western blot analysis of HCT116 cells treated with TNF (0.66 ng/mL). At the indicated time points, whole-cell lysates were prepared and equal amounts of proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with specific indicated antibodies. Anti-GAPDH antibody was used as a loading control. Experiments were repeated 3 times, and representative images are shown.

FIGURE 5

MiR-26b targets DIP1 in vitro and in vivo. A, miR-26b sequences in the 3′-UTR of human DIP1 mRNA as predicted by TargetScan (v.5.1). The miR-26b seed sequence and its predicted binding site in the DIP1 3′-UTR are shown underlined. B, HCT116 cells were cotransfected with the vectors containing the full-length 3′-UTR, mutated 3′-UTR, or an empty pmirGLO vector (250 ng/mL) and an miR-26b mimic (20 nM) or miR scramble (20 nM). Firefly luciferase activity was measured 24 hours later and normalized to Renilla luciferase activity. The experiment was done 3 times in triplicates. Results are expressed as mean ± SD. *P < 0.05 (unpaired 2-tailed t test). C, HCT116 cells were transfected with 20 nM miRNA mimics for 6 hours and then treated with TNF (0.66 ng/mL) for indicated time points. The protein expression was assayed by Western blot using indicated antibodies. Anti-GAPDH antibody was used as a loading control. Band densities were quantified using ImageJ. Experiments were repeated 3 times, and representative images are shown. D, Relative expression of DIP1 mRNA was determined by qRT-PCR from biopsy specimens of healthy cohort (n = 10) and patients with active UC (n = 10). In 5 healthy samples, DIP1 mRNA was not amplifiable. Results were normalized using GAPDH expression level and performed at least 3 times in triplicates.

FIGURE 6

IPA-assisted network analysis of miR-26b target genes/DAPK interaction partners. A, IPA toxicology report. B, IPA: canonical pathways, diseases, and biofunctions. C, Comparative pathway analysis: overlay of potential target genes involved in apoptosis, proliferation, and inflammation.