Crohn's disease (CD) patients present characteristic abnormalities in the mesenterial adipose tissue (MAT) near the affected intestinal area. The MAT is thickened and wraps around the bowel circumference.(1) Recent evidence indicates that this tissue plays a role in storing memory immune cells and potentially supporting antigen-driven immune responses.(2) Therefore, the present study aimed to identify the microRNAs (miR) pattern in the MAT of CD patients by means of an RNA sequencing (RNAseq) analysis (2) and to perform a biological validation of the results comparing to controls (CTR).
For this purpose, 25 patients with active ileocecal CD who underwent surgery were included in the study. The CTR group consisted of 15 patients operated on for other diseases, except inflammatory bowel diseases. The in silico analysis of the modulated miR was performed by TargetScan and the enrichment of the metabolic pathways through the DAVID platform. The biological validation of the transcripts was performed by RT-qPCR analysis. The data were analyzed using the nonparametric Mann-Whitney Test. Univariate and multivariate analysis were performed based on the Cox regression model for correlations between gene expressions and the disease recurrence after surgery. The level of significance was set at P < 0.05. The study was approved by the Research Ethics Committee.
RNAseq identified a significant increase in miR-650 expression in the MAT of the CD group compared to the CTR (FC = 23.45; P < 0.01). Among the 227 downregulated genes, 25 were validated by in silico analysis as a predicted target for miR-650. The enrichment analysis of metabolic pathways containing the miR-650 target genes identified the metabolism pathway of alanine, aspartate and glutamate. GFPT2 and ALDH4A1 were identified as miR-650 target genes of this enriched pathway. The biological validation by RT-qPCR confirmed significant increased miR-650 expression in the MAT of CD compared to the CTR (P = 0.03), besides decreased levels of GFPT2 (P = 0.026) and ALDH4A1 (P = 0.0063) target genes. Moreover, Cox regression analysis showed that the miR-650 levels in the MAT of CD patients strongly correlated with the post-operative disease recurrence in the first 36 postoperative months (Hazard Ratio = 6.85; Confidence Interval 95%; P = 0.006).
For the first time, the modulation of miR-650 and its target genes (ALDH4A1 and GFPT2) were validated in the MAT of CD patients. Indeed, the miR-650 levels correlated to a higher risk of postoperative disease recurrence. Although a larger multicenter prospective study is needed, these findings may constitute a potential tool to guide the clinical management after surgical resection.
São Paulo Research Foundation (FAPESP) #2018/05584-4.
1. Coffey JC, O'Leary DP. The mesentery: Structure, function, and role in disease. Lancet Gastroenterol Hepatol 2016;1:238–47.
2. Silva FAR, et al. Whole transcriptional analysis identifies markers of B, T and plasma cell signaling pathways in the mesenteric adipose tissue associated with Crohn's disease. J Transl Med 2020;18:44.