Colonic epithelial miR-31 associates with the development of Crohn's phenotypes

Abstract

Background: Crohn's disease (CD) is highly heterogeneous, due in large part to variability in cellular processes that underlie the natural history of CD, thereby confounding effective therapy. There is a critical need to advance understanding of the cellular mechanisms that drive CD heterogeneity.

Methods: We performed small RNA sequencing of adult colon tissue from CD and NIBD controls. Colonic epithelial cells and immune cells were isolated from colonic tissues, and microRNA-31 (miR-31) expression was measured. miR-31 expression was measured in colonoid cultures generated from controls and patients with CD. We performed small RNA-sequencing of formalin-fixed paraffin-embedded colon and ileum biopsies from treatment-naive pediatric patients with CD and controls and collected data on disease features and outcomes.

Results: Small RNA-sequencing and microRNA profiling in the colon revealed 2 distinct molecular subtypes, each with different clinical associations. Notably, we found that miR-31 expression was a driver of these 2 subtypes and, further, that miR-31 expression was particularly pronounced in epithelial cells. Colonoids revealed that miR-31 expression differences are preserved in this ex vivo system. In adult patients, low colonic miR-31 expression levels at the time of surgery were associated with worse disease outcome as measured by need for an end ileostomy and recurrence of disease in the neoterminal ileum. In pediatric patients, lower miR-31 expression at the time of diagnosis was associated with future development of fibrostenotic ileal CD requiring surgeryCONCLUSIONS. These findings represent an important step forward in designing more effective clinical trials and developing personalized CD therapies.

Funding: This work was supported by CCF Career Development Award (SZS), R01-ES024983 from NIEHS (SZS and TSF), 1R01DK104828-01A1 from NIDDK (SZS and TSF), P01-DK094779-01A1 from NIDDK (SZS), P30-DK034987 from NIDDK (SZS), 1-16-ACE-47 ADA Pathway Award (PS), UNC Nutrition Obesity Research Center Pilot & Feasibility Grant P30DK056350 (PS), CCF PRO-KIIDS NETWORK (SZS and PS), UNC CGIBD T32 Training Grant from NIDDK (JBB), T32 Training Grant (5T32GM007092-42) from NIGMS (MH), and SHARE from the Helmsley Trust (SZS). The UNC Translational Pathology Laboratory is supported, in part, by grants from the National Cancer Institute (3P30CA016086) and the UNC University Cancer Research Fund (UCRF) (PS).

Keywords: Gastroenterology; Genetics; Inflammatory bowel disease.

Figure 1. Two distinct molecular subtypes across multiple data types in adult Crohn’s disease (CD).

Principal components analysis (PCA) of microRNA (A) and long noncoding RNA (B) expression profiles for patients with CD and patients with NIBD exhibit (black, n = 11–12) distinct clusters; 1 enriched for colon-like CD patients (blue, n = 9–11), and another enriched for ileum-like CD patients (red, n = 9–10). Supplemental Tables 1 and 2 provide the PCA loadings for the above plots.

Figure 2. miR-31 is a driver of colon-like and ileum-like stratification.

(A) Principal components analysis of miRNA expression data from small RNA-seq. miR-31 expression (blue–gold, low–high) appears to distinguish the NIBD (n = 12) and colon-like samples (n = 9) from ileum-like samples (n = 9). (B) Normalized miR-31 expression exhibits a significant upregulation in Crohn’s disease subgroups (colon-like, n = 9; ileum-like, n = 9) samples compared with NIBD samples (n = 12). (C) UCSC browser representation of normalized RNA-seq reads mapping to the miR-31 host gene for NIBD colon (black, n = 11), colon-like CD (blue, n = 9), NIBD ileum (purple, n = 1), and ileum-like CD (red, n = 9). miR-31 transcript expression levels from small RNA-seq (RPMMM) are displayed to the right of each track. FDR adjusted P values determined using DESeq2, with data presented as mean RPMMM ± SEM.

Figure 3. miR-31 is specifically upregulated in intestinal epithelial cells.

RT-qPCR for miR-31 (A), APOA1 (B), and CEACAM7 (C) in an independent adult cohort displays colon-like (blue; n = 27–28) and ileum-like (red; n = 12) clustering patterns for CD samples compared with NIBD samples (black; n = 18–29). (D) RT-qPCR of 5 colon-specific cell types reveal significant miR-31 upregulation in intestinal epithelial cells isolated from CD patients (n = 11–20) relative to NIBD controls (n = 8–16). (Eight NIBD matched and 6 CD matched across all cell types). (E) Relative miR-31 expression by qPCR of colonoid cultures generated from NIBD controls (n = 4) compared with CD patients (n = 4). miR-31 expression is increased in fresh crypts and remains higher at day 2 and day 6 of colonoid culture. miRNA levels are relative to RNU-48 expression compared with fresh NIBD crypts. Significance values determined by a 2-tailed unpaired Student’s t test. Data are presented as mean ± SEM.

Figure 4. miR-31 is differentially expressed in treatment-naive pediatric Crohn’s disease (CD) samples.

miR-31 expression is significantly upregulated in the colon (A) and ileum (B) of treatment-naive pediatric CD samples (colon, n = 76; ileum, n = 60) compared with pediatric NIBD samples (colon, n = 48; ileum, n = 50). Principal components analysis of miRNA expression profiles from small RNA-seq results in distinct clusters of NIBD and CD patients for colon (C) and ileum (D) samples. Points are colored according to miR-31 expression (blue–gold, low–high). Significance determined by a 2-tailed unpaired Student’s t test where P < 0.05. Data presented as mean ± SEM.