Modulation of faecal metagenome in Crohn's disease: Role of microRNAs as biomarkers

Abstract

Background: The gut microbiota plays a key role in the maintenance of intestinal homeostasis and the development and activation of the host immune system. It has been shown that commensal bacterial species can regulate the expression of host genes. 16S rRNA gene sequencing has shown that the microbiota in inflammatory bowel disease (IBD) is abnormal and characterized by reduced diversity. MicroRNAs (miRNAs) have been explored as biomarkers and therapeutic targets, since they are able to regulate specific genes associated with Crohn's disease (CD). In this work, we aim to investigate the composition of gut microbiota of active treatment-naïve adult CD patients, with miRNA profile from gut microbiota.

Aim: To investigate the composition of gut microbiota of active treatment-naïve adult CD patients, with miRNA profile from gut microbiota.

Methods: Patients attending the outpatient clinics at Valme University Hospital without relevant co-morbidities were matched according to age and gender. Faecal samples of new-onset CD patients, free of treatment, and healthy controls were collected. Faecal samples were homogenized, and DNA was amplified by PCR using primers directed to the 16S bacterial rRNA gene. Pyrosequencing was performed using GS-Junior platform. For sequence analysis, MG-RAST server with the database Ribosomal Project was used. MiRNA profile and their relative abundance were analyzed by quantitative PCR.

Results: Microbial community was characterized using 16S rRNA gene sequencing in 29 samples (n = 13 CD patients, and n = 16 healthy controls). The mean Shannon diversity was higher in the healthy control population compared to CD group (5.5 vs 3.7). A reduction in Firmicutes and an increase in Bacteroidetes were found. Clostridia class was also significantly reduced in CD. Principal components analysis showed a grouping pattern, identified in most of the subjects in both groups, showing a marked difference between control and CD groups. A functional metabolic study showed that a lower metabolism of carbohydrates (P = 0.000) was found in CD group, while the metabolism of lipids was increased. In CD patients, three miRNAs were induced in affected mucosa: mir-144 (6.2 ± 1.3 fold), mir-519 (21.8 ± 3.1) and mir-211 (2.3 ± 0.4).

Conclusion: Changes in microbial function in active non-treated CD subjects and three miRNAs in affected vs non-affected mucosa have been found. miRNAs profile may serve as a biomarker.

Keywords: Bacteroidetes; Crohn’s disease; Dysbiosis; Firmicutes; microRNAs.

Figure 1

Ecological and metagenomic analysis. A: Number of readings for each sample using the 16S massive sequencing technique in GS Junior. See methods section for details. B: Principal components analysis. The control samples and Crohn’s disease are observed in well-defined groups. The data was selected with the Ribosomal Project database using a maximum e-value of 10^-5, a minimum identity of 75%, and a minimum length alignment of 15 bp. PC: Principal components; CD: Crohn’s disease sample.

Figure 2

Box plot showing a significant difference in the Clostridia class between control group (blue) and Crohn’ disease (green).

Figure 3

Bar graph with the mean of each group (Crohn’s disease and control population) and family taxon (95% confidence level).

Figure 4

Bar chart with the mean of each taxonomic group (gender) according to group (control vs Crohn’s disease) and differences with 95% confidence level.

Figure 5

Functional analysis of the microbiota. Significant differences (P < 0.05) in biosynthesis and glycan metabolism, carbohydrate metabolism, lipid metabolism, catabolism, digestive system, amino acid metabolism and immune system were found. C: Control sample; CD: Crohn’s disease sample.

Figure 6

Three microRNAs were found increased in samples from patients with Crohn’s disease. Individual microRNA levels in 10 patients with Crohn’s disease are represented. miRNA: MicroRNA.