The microRNA let-7b-5p Is Negatively Associated with Inflammation and Disease Severity in Multiple Sclerosis

Abstract

The identification of microRNAs in biological fluids for diagnosis and prognosis is receiving great attention in the field of multiple sclerosis (MS) research but it is still in its infancy. In the present study, we observed in a large sample of MS patients that let-7b-5p levels in the cerebrospinal fluid (CSF) were highly correlated with a number of microRNAs implicated in MS, as well as with a variety of inflammation-related protein factors, showing specific expression patterns coherent with let-7b-5p-mediated regulation. Additionally, we found that the CSF let-7b-5p levels were significantly reduced in patients with the progressive MS compared to patients with relapsing-remitting MS and were negatively correlated with characteristic hallmark processes of the two phases of the disease. Indeed, in the non-progressive phase, let-7b-5p inversely associated with both central and peripheral inflammation; whereas, in progressive MS, the CSF levels of let-7b-5p negatively correlated with clinical disability at disease onset and after a follow-up period. Overall, our results uncovered, by the means of a multidisciplinary approach and multiple statistical analyses, a new possible pleiotropic action of let-7b-5p in MS, with potential utility as a biomarker of MS course.

Trial registration: ClinicalTrials.gov NCT03217396.

Keywords: Expanded Disability Status Scale (EDSS); G_CSF; IL5; RANTES; inflammation; let-7; miRNAs; multiple sclerosis (MS); progressive multiple sclerosis (PMS).

Conflict of interest statement

D.C. is the recipient of an Institutional grant from Biogen. No personal compensation was received. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. F.B. acted as Advisory Board members of Teva and Roche and received honoraria for speaking or consultation fees from Merck Serono, Teva, Biogen Idec, Sanofi, and Novartis and non-financial support from Merck Serono, Teva, Biogen Idec, and Sanofi. R.F. received honoraria for serving on scientific advisory boards or as a speaker from Biogen, Novartis, Roche, and Merck and funding for research from Merck. M.S. received research support and consulting fees from Biogen, Merck-Serono, Novartis, Roche, Sanofi, Teva. D.C. is an Advisory Board member of Almirall, Bayer Schering, Biogen, GW Pharmaceuticals, Merck Serono, Novartis, Roche, Sanofi-Genzyme, and Teva and received honoraria for speaking or consultation fees from Almirall, Bayer Schering, Biogen, GW Pharmaceuticals, Merck Serono, Novartis, Roche, Sanofi-Genzyme, and Teva. He is also the principal investigator in clinical trials for Bayer Schering, Biogen, Merck Serono, Mitsubishi, Novartis, Roche, Sanofi-Genzyme, and Teva. His preclinical and clinical research was supported by grants from Bayer Schering, Biogen Idec, Celgene, Merck Serono, Novartis, Roche, Sanofi-Genzyme and Teva. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The other authors declare that they have no competing interests.

Figures

Figure 1

Let-7 family is a good candidate as a MS-associated miRNA. (A) DNA sequences alignment of the mature miRNAs part of the let-7 family. In black the nucleotides positions conserved among all members of the let-7 family. The seed sequence is indicated as a red box. (A’) Genomic organization of let-7 family genes in humans. Gene clusters are divided according to the genome organization (intronic or intergenic). Chromosome strands are indicated by (+) or (−). Figure information sourced from http://microrna.sanger.ac.uk/sequences/. (B) Functional analysis of experimentally validated target mRNAs of let-7 obtained from miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php). The most represented Gene Ontology categories for target mRNAs are reported in the figure. Size dots are correlated with the number of genes that belong to a Gene Ontology category and dots are colored according to the Benjamini-Hochberg false discovery rate adjusted p-values from blue (higher p-adjusted) to red (lower p-adjusted). (B’) Network of let-7 targets that can be ascribed to three main processes involved in MS pathophysiology: inflammation (light blue rectangle); neuronal homeostasis (green rectangle); RNA metabolism (orange rectangle). Target mRNAs of let-7 involved in more than one process are represented into the rectangle overlapping zones. Targets participating in other pathways are grouped into a light violet rectangle (26 out of 130).

Figure 1

Let-7 family is a good candidate as a MS-associated miRNA. (A) DNA sequences alignment of the mature miRNAs part of the let-7 family. In black the nucleotides positions conserved among all members of the let-7 family. The seed sequence is indicated as a red box. (A’) Genomic organization of let-7 family genes in humans. Gene clusters are divided according to the genome organization (intronic or intergenic). Chromosome strands are indicated by (+) or (−). Figure information sourced from http://microrna.sanger.ac.uk/sequences/. (B) Functional analysis of experimentally validated target mRNAs of let-7 obtained from miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php). The most represented Gene Ontology categories for target mRNAs are reported in the figure. Size dots are correlated with the number of genes that belong to a Gene Ontology category and dots are colored according to the Benjamini-Hochberg false discovery rate adjusted p-values from blue (higher p-adjusted) to red (lower p-adjusted). (B’) Network of let-7 targets that can be ascribed to three main processes involved in MS pathophysiology: inflammation (light blue rectangle); neuronal homeostasis (green rectangle); RNA metabolism (orange rectangle). Target mRNAs of let-7 involved in more than one process are represented into the rectangle overlapping zones. Targets participating in other pathways are grouped into a light violet rectangle (26 out of 130).

Figure 2

Let-7b-5p is a hub in the network of miRNAs in the CSF of patients with MS. (A) Heat map of Pearson’s correlation coefficients (r) between 23 miRNAs (relative to miR-204-5p, according to the ΔCt calculation) detected by qPCR in the CSF of the main cohort of patients (n = 166) at T0. In the upper triangle, r values of significant correlation (p < 0.05) were represented by coloured circles according to the scale (r > 0 is positive correlation, 0 no correlation and <−1 is negative correlation). In the lower triangle, r values are reported following the color code. Squares represent three different clusters identified by hierarchical clustering using the cutree R function with k = 3. Only statistically significant correlations with FDR < 0.05 are shown. (A’) Network representation of miRNA correlation. In red, there are detected members of the let-7 family (let-7b-5p, let-7e-5p and let-7f-5p). Blue nodes are other miRNAs relevant for MS, which correlate each other and/or with let-7b-5p (r ≥ 0.5). In green are miRNAs with r < 0.5. Pink and light blue areas represent, respectively, the first and the second correlation clusters, highlighted in panel A of the figure by the two lower squares.

Figure 3

Let-7b-5p is an anti-inflammatory regulator of the complex pathway of soluble biochemical factors circulating in the MS CFS. By means of hierarchical cluster analysis, we divided in homogeneous groups, the 27 inflammation-related proteins (variables), quantified by multiple assays on 166 MS patients at T0. (A) We used the silhouette method to identify the optimal number of clusters, equal to two main clusters (A’) The result of this analysis was represented as a dendrogram: cluster 1 (red) with 7/27 inflammatory proteins (IFNγ, IL1ra, IL8, IP10, IL5, G_CSF, RANTES); cluster 2 (blue) including 20/27 inflammatory proteins (PDGFbb, IL12_p70, FGFbasic, IL15, IL2, GM_CSF, IL17, IL13, IL4, IL9, IL7, IL6, VEGF, IL1β, IL10, MCP1, eotaxin, TNFα, MIP1a, MIP1b).

Figure 4

The levels of let-7b-5p are different according to diverse MS disease subtypes. (A) Box and whisker plots of let-7b-5p levels in the CSF, isolated from control subjects (Ctr) compared to all MS patients (Ctr, n = 20; All patients, n = 166; Mann-Whitney test, p > 0.05). (A’) Box and whisker plots of let-7b-5p levels in the CSF isolated from control subjects compared to patients separated in CIS/RIS, RRMS and PMS patients (Ctr, n = 20; CIS/RIS, n= 25; RRMS, n = 117; PMS, n = 24; Kruskal-Wallis test, * p < 0.05 RRMS vs. PMS). Data were normalized to miR-204-5p expressed as 2−ΔCt let7b-5p-miR-204-5p). Values are median of 2−ΔCt with 10–90% percentiles (error bars) and 25–75% percentiles (open boxes).

Figure 5

The correlations with inflammation and cognitive performances revealed a putative protective role of let-7b-5p in non-progressive phase. (A) Correlation plot between let-7b-5p levels and the count of peripheral T cells of non-PMS (n = 140) at T0. A negative correlation was observed (Spearman’s correlation: rs = −0.216, * p < 0.01). (B,B’) Correlation plot between let-7b-5p levels and Scheme 106. and Phonemic (n = 95) verbal fluency of non-PMS patients at T0. A positive correlation was observed in both executive and categorical memory functions (Spearman’s correlation, B: rs = −0.294, p < 0.01; B’: rs = 0.218, ** p < 0.05).

Figure 6

The CSF level of let-7b-5p correlates with disease severity in MS progressive phase. (A) Correlation plot between let-7b-5p levels and the count of peripheral T cells of PMS patients (n = 22, A) at T0. (B,B’) Correlation plot between let-7b-5p levels and EDSS of PMS patients at T0 (n = 24, B) and after a follow-up period ((Tf1), n = 21, B’). A negative correlation was observed at both T0 (Spearman’s correlation, Spearman’s r = −0.463, * p < 0.05) and Tf1 (Spearman’s correlation, Spearman’s r = −0.536, * p < 0.05).