Introduction
Inflammatory bowel diseases (IBD) are a distinct class of gastrointestinal disorders mainly represented by Crohn’s Disease (CD) and Ulcerative Colitis (UC). The dysregulation of microRNAs (miRNAs), a class of conserved endogenous, small non-coding RNA molecules, both tissue-derived and circulating, have shown to be promising biomarkers in IBD. These biomarkers can be useful for differential diagnosis, drug response prediction and can have prognostic value for the evolution of the disease. Moreover, miRNAs are interesting tools for potential future therapeutic approaches by miRNA antagonists or mimics [1].
Aims & Methods
In this study we aimed to perform a preliminary screening of a large panel of 179 miRNAs in the plasma of 6 patients with IBD and 12 non-IBD patients (CTRL) in order to identify a set of highly dysregulated miRNAs to be investigated in a larger cohort. Isolation of miRNAs has been conducted using miRNeasy Serum/Plasma Kit and the miRNAs expression has been evaluated using the Human serum/plasma focus, MIRCURY LNA miRNA Focus PCR panel (Qiagen) by qRT-PCR. After excluding 29 miRNAs that were not expressed in the investigated samples, we identified miR-19a-3p and miR-17-5p as the most stable miRNAs by the RefFinder analysis. Thus, the Ct values of the other 148 miRNAs have been normalized against the geometric mean of these two miRNAs as endogenous controls using the 2-DCt method. All the clinical, sociodemographic and miRNAs expression data have been analyzed using SPSS. Categorical variables were tested by means of the chi-square test, and continuous variables with t-test or the non-parametric Man Whitney test, according to the normality of the data distribution.
Results
The two groups were age matched (mean age IBD= 45.83±21.27, mean age CTRL = 50.08±8.16, p=0.543) and did not differ for sex distribution (IBD= 33.3% female, CTRL=58.3% female, χ2= 1.00 and p=0.317). The statistical analysis revealed 45 miRNAs differentially expressed (p<0.05). In particular, we identified 29 overexpressed miRNAs (miR-223-3p, miR-143-3p, let-7f-5p, miR-30b-5p, miR-26a-5p, let-7a-5p, miR-142-3p, miR-374a-5p, miR-339-5p, miR-150-5p, miR-454-3p, let-7d-5p, miR-29a-3p, miR-146a-5p, miR-199a-3p, miR-221-3p, miR-142-5p, miR-191-5p, miR-338-3p, let-7g-5p, miR-103a-3p, miR-23a-3p, miR-145-5p, miR-374b-5p, miR-24-3p, miR-107, miR-27b-3p, let-7c-5p, miR-26b-5p) in the IBD group compared with the CTRL showing a fold regulation (FR)>1.5 and 7 miRNAs underexpressed reporting a FR<-1.5 (miR-421, miR-320d, miR-106b-3p, miR-320c, miR-20b-5p, miR-320b, miR-486-5p).
Notably, the expression of let-7a-5p that was upregulated in the IBD group (FR=2.45; p=0.001) positively correlated with the years of disease duration (p=0.01, Pearson r=0.919). The same positive correlation was observed for miR-30b-5p (p=0.049, Pearson r=0.812).
Conclusion
The reported data, showing 45 miRNAs differentially expressed in the plasma of IBD patients compared to controls, represent preliminary results that must be validated. The most statistically significant dysregulated miRNAs, as well as those that correlated with the years of disease duration and other clinical parameters will be investigated in a large IBD cohort in order to examine the potential value of these miRNAs as diagnostic and prognostic biomarkers.
This investigation received financial support from ICGEB (Contract No. CRP/22/019, ICGEB Ref. No. CRP/ROU21-01)
References
1. James JP, Riis LB, Malham M, Høgdall E, Langholz E, Nielsen BS. MicroRNA Biomarkers in IBD-Differential Diagnosis and Prediction of Colitis-Associated Cancer. Int J Mol Sci. 2020 Oct 24;21(21):7893. doi: 10.3390/ijms21217893. PMID: 33114313; PMCID: PMC7660644.