Table 1.
MicroRNAs in blood of human patients with MS
| Authors, country | Number of patients, gender, ages | Comparison | Changes in miRNAs in DR patients | Functional outcomes | Conclusion | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Peripheral blood | ||||||||||||||
| Nuzziello et al., 2018; Italy | 58 MS patients with adult onset of the disease (AOMS), 16 M/42 F, 37.8 ± 11.3 yrs, disease duration 13.4 ± 9.3 yrs, MS course 54 RR/4 SP/0 PP, EDSS 2.7 ± 1.1, DMT 56 Y/2 N. Peripheral blood samples were collected and frozen at –20oC. | 20 HC, 6 M/14 F, 43.2 ± 3.1 yrs. They were recruited among volunteers who did not show clinical signs or instrumental evidences of inflammatory or neurological diseases, and who had negative family histories for MS and other neurodegenerative diseases. | The 13 miRNAs that were differentially expressed in pediatric MS were analyzed by qPCR in the AOMS patients. Six miRNAs (miR-320a, miR-125a-5p, miR-652-3p, miR-185-5p, miR-942-5p, miR-25-3p) were significantly dysregulated in AOMS compared to HC. The qPCR analysis showed statistically significant upregulation of miR-320a (fold change, FC = 1.79), miR-125a-5p (FC = 1.89), miR-652-3p (FC = 1.51), miR-185-5p (FC = 1.5), miR-942-5p (FC = 1.67), and miR-25-3p (FC = 1.49) in AOMS compared to HC. | By ROC analysis, AUC values for the validated miRNAs ranged from 0.701 to 0.735 for miR-320a, miR-185-5p, miR-125a-5p and miR-652-3p, discriminating AOMS patients from HC and with miR-320a having the best AUC of 0.735. Using databases containing experimentally validated miRNA-target interactions (miRtarbase and DIANA-Tarbase), 155 miRNA-target pairs were validated by reporter gene assays. Several target genes (TP53, SLC4A10, CDKN1A, EKBB2, ATRX, ST6GAL2, PTEN, FAM160B1, SMAD7, IKZF4, PHLPP2, MCL1, KCNS3, NFATC3, AR, IGF1R, PCDHA4, TANC2, ZNF704, WWC2, NTRK3, NCAN, VEGFA, MSI1, LCOR, and RBM20) were shared by two of the following miRNAs: miR-125a-5p, miR-320a, miR-25-3p, and miR-185-5p. | MiR-320a, miR-125a-5p, miR-652-3p, miR-185-5p, miR-942-5p, miR-25-3p were significantly upregulated in AOMS and may be considered as circulating biomarkers of the disease. | |||||||||
| Liguori et al., 2018; Italy | 19 patients with pediatric MS (PedMS), 9 M/10 F, 15.5 ± 2.7 yrs, disease duration 2.8 ± 3.3 yrs, MS course RR, EDSS 2.7 ± 1.1, DMT 9 Y/ 10 N, education 9.6 ± 2.5 yrs, cognitive abilities 7 CI/10 CP/2 NA. Peripheral blood samples were collected and frozen at –20oC. At the time of blood sample, all treated patients were under interferon β-1 therapy. PedMS patients were analyzed during the inactive phase of a RR course of the disease (no clinical relapses or steriod assumption within 30 days of study entry, no Gd-enhancing lesions at the concomitant MRI), and were steriod-free (at least 1 mth prior to blood sample). 9 Ped MS patients had taken interferon β-1a for 21–673 days from blood sample. | 20 healthy pediatric controls (PC), 14 M/6 F, 8.8 ± 3.3 yrs, education 3.4 ± 2.6 yrs, cognitive abilities NA. | The PedMS patients and PC controls differed significantly for age at study entry, but there was no significant gender difference between them. The investigation performed using a High-Throughput Next Generation Sequencing approach was followed by an integrated bioinformatics/ biostatistics analysis. This revealed 49 mature miRNAs significantly differentially expressed between PedMS and PC. After further selection, the total was reduced to 27 miRNAs that were subjected to qRT-PCR validation. In the validation study, 12 miRNAs (let-7a-5p, let-7b-5p, miR-25-3p, miR-125a-5p, miR-942-5p, miR-221-3p, miR-652-3p, miR-182-5p, miR-185-5p, miR-181a-5p, miR-320a, miR-99b-5p) were significantly upregulated and 1 miRNA (miR-148b-3p) significantly downregulated in PedMS compared to PC. | The interactions between the validated miRNAs and their targets uncovered predicted genes related to immunological functions (i.e., TNFSF13B, TLR2, BACH2, KLF4), as well as genes involved in autophagy-related processes (i.e., ATG16L1, SORT1, LAMP2) and ATPase activity (i.e., ABCA1, GPX3). No significant molecular profiles were associated with any PedMS demographic/clinical features. Both miRNAs and mRNA expressions predicted the PedMS and PC phenotypes with an accuracy of 92% and 91%, respectively. | 12 miRNAs (let-7a-5p, let-7b-5p, miR-25-3p, miR-125a-5p, miR-942-5p, miR-221-3p, miR-652-3p, miR-182-5p, miR-185-5p, miR-181a-5p, miR-320a, miR-99b-5p) were significantly upregulated and 1 miRNA (miR-148b-3p) significantly downregulated in PedMS compared to PC. | |||||||||
| Peripheral blood leukocytes (PBL) | ||||||||||||||
| Magner et al., 2016; USA | 24 MS patients on no therapy, 2 M/22 F, 22 were Caucasians, 52 ± 11 yrs, disease course 19 RRMS/5 SPMS, disease duration 16.9 ± 11.5 yrs, EDSS 3.5 ± 1.6. All patients were evaluated clinically within 1 month of their study participation and assessed using EDSS scale. MS patient scores 1.0–4.5 and 5.0–6.5 indicate degrees of impaired ambulation. Patients with scores ≥ 7 are not ambulatory. Whole blood samples taken at 0 and 48 hours were separated over Ficoll and recovered leukocytes were lysed for total RNA. | 25 HC, 5 M/20 F, 25 were Caucasians, 50 ± 9 yrs | Untreated MS patient PBL Dicer protein levels were 40% lower than the levels found in age and gender matched HC (P = 0.049). Further analysis suggested that Dicer protein levels decreased with disease progression. Despite the significant changes in Dicer protein levels, there were no statistically significant difference in DICER1 mRNA levels between untreated MS and HC. By next generation sequencing analysis, significant differences in miRNA expression levels were found for untreated RRMS patients compared to HC: miR-484, miR-9-5p, miR-323b-3p, miR-15b-5p, miR-145-5p, miR-337-5p, miR-485-5p, miR-654-3p, miR-377-3p, miR-493-5p, miR-301b, miR-409-3p, miR-494 (all upregulated), miR-451a, miR-144-5p, miR-101-3p (all downregulated) with false discovery rates 0.0019–0.0462. The highest logFC values were for miR-337-5p (logFC = 1.58), miR-144-5p (logFC = –1.98) and miR-451a (logFC = –1.71). In untreated RRMS patients, two miRNAs (miR-146b-5p, miR-874) were negatively correlated with EDSS and two others (miR-107, miR-3614) were positively correlated. MiR-107 has been shown to target Dicer (Asirvatham et al., 2008; Martello et al., 2010). | Potential biomarkers for untreated RRMS were identified. Analysis of PBL miRNA will include miRNA expressed in each circulating cell population as well as miRNA shed from tissues, often in exosomes, that become associated with PBL. | ||||||||||
| Blood serum | ||||||||||||||
| Vistbakka et al., 2018; Finland | 53 RRMS patients, 12 M/41 F, 35.3 ± 7.1 yrs, disease duration 5.4 ± 5.4 yrs, EDSS 1.6 ± 1.7, progression index 0.21 ± 0.50; 20 PPMS patients, 10 M/10 F, 52.7 ± 8.0 yrs, disease duration 7.6 ± 6.5 yrs, EDSS 5.4 ± 1.8, progression index 0.38 ± 0.82. Diagnosis of MS was based on the revised McDonald criteria. Active DMT treatment was ongoing in 37 patients with RRMS. All patients underwent clinical and neurological examination including assessment of neurological disability expressed by EDSS score. Progression index was calculated by dividing EDSS score by disease duration from diagnosis. An average annual relapse rate (ARR) was calculated in RRMS group starting from the time of diagnosis. Prior to sampling, none of the patients had any relapses for at least 8 weeks. The mean ARR was 1.7 ± 3.4, and mean number of relapses during the last 2 yrs was 0.9 ± 1.1. Venous blood was collected and sera separated by centrifugation (1600 × g, 15 minutes, room temperature) and stored at –80oC. | 27 HC, 9 M/18 F, 38.2 ± 11.8 yrs. Healthy controls had no history of any autoimmune disease or use of any immunomodulatory therapy. | By RT-PCR, miR-24-3p and miR-191-5p were expressed in all the samples, while miR-128-3p and miR-376c-3p were expressed in 95% and 87% of the samples, respectively. Overexpression of miR-128-3p, miR-191-5p, and miR-24-3p occurred in sera of patients with MS (RRMS and PPMS) compared to HC, although after the Bonferroni correction (to reduce type-1 errors, false positives) only miR-191-5p and miR-24-3p remained statistically significant. Expression levels of miR-376c-3p did not differ between the groups. Overexpression was found of miR-191-5p (RRMS, P = 0.01, FC = 1.65; PPMS, P < 0.001, FC = 1.75) and miR-24-3p (RRMS, P = 0.01, FC = 2.10; PPMS, P = 0.01, FC = 3.58) in both subtypes and miR-128-3p in PPMS (P = 0.03, FC = 1.72) compared to HC. After the Bonferroni correction, miR-128-3p did not reach statistical significance. No statistically significant differences were found between RRMS and PPMS. Comparison between untreated and treated MS patients did not show any statistical differences in miRNA expression levels. In the male population, miR-128-3p and miR-191-5p were overexpressed in RRMS (P = 0.007, FC = 11.33 and P = 0.009, FC = 4.68), PPMS (P = 0.023, FC = 3.56 and P = 0.022, FC = 4.01) and among all patients with MS (P = 0.003, FC = 7.49 and P = 0.004, FC = 4.40) compared to HC. Also, miR-128-3p was overexpressed in RRMS compared to PPMS (P = 0.038, FC = 3.18). However, the results of PPMS to HC and PPMS to RRMS comparisons did not pass the Bonferroni correction. In the female population, miR-24-3p was overexpressed in RRMS (P = 0.020, FC = 1.78), PPMS (P = 0.011, FC = 6.06) and in all patients with MS (P = 0.007, FC = 2.62) compared to HC. The RRMS to HC comparison did not pass the Bonferroni correction. Overexpression of miR-24-3p was detected in PPMS cohort in male to female comparison (P = 0.038, FC = 8.17) but it did not pass the Bonferroni correction. | In the whole MS group, miR-24-3p showed positive correlation with the progression index, and after adjustment for gender, disease subtype, and age, this correlation was preserved. In the RRMS group, miR-128-3p showed positive correlation with ARR, and this became stronger after adjustment for gender and age. In the PPMS group, miR-376c-3p tended to correlate positively with EDSS, but was not preserved after gender and age adjustment. | In MS, serum miR-24-3p and miR-128-3p showed a tendency of association with disability and disease activity, respectively. | |||||||||
| Regev et al., 2018; USA (CLIMB, EPIC) Lebanon (AMIR) Sweden (STOPMS II) | CLIMB cohort: 24 RRMS patients, 5 M/19 F, 32.4 ± 6.6 yrs, disease duration 3.9 ± 2.4 yrs, EDSS 0.6 ± 0.9; 18 SPMS patients, 3 M/15 F, 56.4 ± 9.3 yrs, disease duration 17.2 ± 5.9 yrs, EDSS 5.5 ± 1.6. AMIR cohort: 31 RRMS patients, 10 M/21 F, 33.5 ± 10.2 yrs, disease duration 4.6 ± 4.7 yrs, EDSS 2.1 ± 1.2; 5 SPMS patients, 3 M/2 F, 40.2 ± 7.8 yrs, disease duration 15.2 ± 11.0 yrs, EDSS 4.5 ± 1.4. EPIC cohort: 27 RRMS patients, 7 M/20 F, 37.6 ± 6.8 yrs, disease duration 3.9 ± 2.4 yrs, EDSS 1.0 ± 0.8; 7 SPMS patients 0 M/7 F, 52.7 ± 5.8 yrs, disease duration 19.0 ± 6.0 yrs, EDSS 5.1 ± 1.3. STOPMS II cohort: 33 RRMS patients, 10 M/23 F, 33.1 ± 9.4 yrs, disease duration 2.2 ± 3.4 yrs, EDSS 1.7 ± 1.7; 21 SPMS patients, 10 M/11 F, 43.2 ± 4.3 yrs, disease duration 19.6 ± 5.2 yrs, EDSS 5.3 ± 1.5. None of the selected patients were receiving DMT at the time of sample collection. Blood samples were collected in tubes without additives and kept at room temperature for 30–60 minutes. Each sample was centrifuged (2000 r/min, 10 minutes) to separate the serum and then stored at –70oC. | CLIMB cohort: 30 healthy controls (HC), 5 M/25 F, 45.4 ± 15.0 yrs; AMIR cohort: 19 HC, 7 M/12 F, 37.6 ± 11.2 yrs; EPIC cohort: 26 HC, 14 M/12 F, 45.5 ± 10.2 yrs; STOPMS II cohort: 13 HC, 7 M/6 F, 30.9 ± 5.6 yrs. | In the reproducibility phase 73 miRNAs showing promise as biomarkers from a larger validation set of participants from CLIMB patients (validation phase) were further analysed by RT-PCR with MS group (24 RRMS, 18 SPMS) and 30 HC from the CLIMB cohort. Five previously validated miRNAs viz. miR-484, miR-320a, miR-320c, miR-486-5p (all increased) and miR-140-5p (decreased) showed a significant difference, and 1 miRNA remained significant after accounting for multiple comparisons to control for false discovery rate (miR-484). In a comparison between RRMS and HC, only miR-484 showed a significant difference after accounting for multiple comparisons. When SPMS and HC were compared, 8 miRNAs were significantly different, and 6 miRNAs (mR-484, miR-140-5p, miR-142-5p, miR-320a, miR-320b, miR-320c) were significantly different after accounting for multiple comparisons. When RRMS and SPMS were compared, no previously validated miRNAs were significantly different between the groups. Four miRNAs, miR-320b, miR-337-3p, miR-199a-5p, and miR-142-5p, were significantly correlated with EDSS in the reproducibility phase cohort. In the transportability phase the 73 miRNAs were further analysed in 3 external cohorts with study participants from 3 MS centers worldwide with 91 RRMS, 33 SPMS and 58 HC. For the comparison of MS and HC, 4 miRNAs were differentially expressed in 2 of the 4 MS cohorts: let-7e-5p, let-7f-5p, miR-486-5p, miR-30e-5p. For the comparison of RRMS and HC, 2 miRNAs were differentially expressed in 2 of the 4 MS cohorts: let-7e-5p and let-7f-5p. For the comparison of SPMS and HC, miR-320a, miR-320b, miR-320c and miR-486-5p were differentially expressed in the CLIMB and STORMS II cohorts. The expression of miR-337-3p negatively correlated with EDSS in the discovery set (n = 85) and validation phase (n = 58). These findings were further tested in samples from the reproducibility phase and transportability phase. Results showed that miR-337-3p was negatively correlated with EDSS in 3 of 4 cohorts. An additional 5 miRNAs (miR-142-5p, miR-199a-5p, miR-330-3p, miR-194-5p, miR-941) showed a significant correlation in 2 of the cohorts. In the CLIMB cohort, SPMS patients have significantly decreased miR-337-3p expression compared to RRMS patients. Comparisons between SPMS and RRMS in the other 3 cohorts showed a similar reduction in miR-337-3p expression in SPMS patients compared to RRMS patients in 3 of the 4 cohorts. | Including let-7c-5p, miR-452-5p and miR-484 in a predictive model for MS vs. HC gave by ROC analysis an AUC value of 0.85 in the validation cohort. | When comparing RRMS with HC, miR-484 was significantly different between the groups after accounting for multiple group comparisons. When SPMS and HC were compared, mR-484, miR-140-5p, miR-142-5p, miR-320a, miR-320b, miR-320c remained significantly different after accounting for multiple group comparisons. In disability correlation analysis, miR-320a, miR-337-3p, miR-199a-5p, and miR-142-5p correlated with the EDSS in the reproducibility phase cohort. SPMS patients had significantly decreased miR-337-3p expression compared to RRMS patients in 3 of the 4 cohorts. | |||||||||
| Wang et al., 2017; China | 30 RRMS patients at relapse, 10 M/20 F, 31.1 ± 13.4 yrs, smoking status 6 Ever/current/24 Never, alcohol consumption 6 Ever/current/24 Never, EDSS 2.8 ± 1.6. Regarding inclusion criteria: clinical diagnoses were confirmed using the 2010 revisions to McDonald diagnostic criteria, all participants were on periods of relapse and Epstein-Barr virus (EBV)-immnoglobulin (Ig)G-positive but EBV-IgM-negative. Regarding exclusion criteria, PPMS and PedMS cases were excluded, patients who met the diagnostic criteria of other systemic autoimmune diseases involving CNS demyelination were excluded, patients who received treatment within the previous 60 days were excluded. Venous blood was collected with no anti-coagulant prior to therapy, allowed to stand for 20 min, centrifuged (1500 × g, 10 minutes, 20oC). Sera was collected and stored at –80oC for analysis. | 30 HC, 10 M/20 F, 34.4 ± 9.4 yrs, smoking status 8 Ever/current/ 22 Never, alcohol consumption 4Ever/current/26Never, EDSS NA. | By qRT-PCR assay, 20 EBV miRNAs were expressed robustly and detectable in all samples. Expression of ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3 was significantly increased in RRMS patients compared to HC (FC = 1.48 and 1.33, respectively), while the other 18 miRNAs showed no significant difference. The upregulated expression of ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3 was associated positively with EDSS scores of RRMS patients | By ROC analysis, the AUC values for ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3 used independently were 0.74 and 0.72, respectively. With a combination of the two miRNAs, the AUC value was 0.76. Among the candidate target genes of ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3, mucosa-associated lymphoid tissue lymphoma transport protein 1 (MALT1) was predicted to have two potential binding sites for ebv-miR-BHRF1-2-5p on the 3′-UTR. MALT1 is involved in immune homeostasis (Gewies et al., 2014; Jaworski et al., 2014). Also a binding site for ebv-miR-BHRF1-3 was found within the mRNA of phosphatase and tensin homologue (PTEN), which had been confirmed previously as a target of ebv-miR-BHRF1-3 (Bernhardt et al., 2016). | Mature EBV miRNAs are transported by exosomes, which protect them from degradation by RNases. Thus, EBV-encoded miRNAs are stable in the serum so that they can be used as a diagnostic marker and monitor of EBV-associated disorders, including MS. Large-scale studies have confirmed that EBV is the only viral agent that is associated positively with MS (Pender et al., 2014; Belbasis et al., 2015). | |||||||||
| Vistbakka et al., 2017; Finland | Screening phase: 18 PPMS patients, 8 M/10 F, 53.5 ± 8.5 yrs, disease duration from first symptoms 15.6 ± 10.0 yrs, disease duration from diagnosis 11.0 ± 7.7 yrs, EDSS 5.9 ± 1.3, progression index 0.8 ± 1.0. Validation phase: 31 PPMS patients, 13 M/18 F, 56.3 ± 9.7 yrs, disease duration from first symptoms 16.8 ± 10.9 yrs, disease duration from diagnosis 10.9 ± 8.4 yrs, EDSS 5.3 ± 1.8, progression index 0.7 ± 0.8; 31 SPMS patients, 7 M/24 F, 48.8 ± 9.5 yrs, disease duration from first symptoms 23.4 ± 8.3 yrs, disease duration from diagnosis 16.6 ± 8.3 yrs, EDSS 5.7 ± 1.4, progression index 0.45 ± 0.3. Blood was collected and allowed to clot for 30 min before centrifugation (1500 × g, 15 minutes). Serum was separated and stored at –80oC. | Screening phase: 10 HC, 4 M/6 F, 51.8 ± 8.2 yrs. Validation phase: 21 HC, 9 M/12 F, 52.7 ± 8.1 yrs. | Expression of miRNAs was measured using RT-PCR. Of the 84 miRNAs analysed in the screening phase, 4 were significantly downregulated (miR-375, miR-130b-3p, miR-141-3p, miR-124-3p) and 5 were upregulated (miR-128-3p, miR-376c-3p, miR-191-5p, miR-26a-5p, miR-24-3p) in PPMS compared to HC. In the validation phase, when the group including all MS patients was compared to HC, 5 miRNAs (miR-128-3p, miR-376c-3p, miR-26a-5p, miR-191-5p, miR-24-3p) were upregulated (FC > 1.5, P < 0.05), but after the Bonferoni correction (to reduce the chances of obtaining false-positive results) only the differences for 4 miRNAs (miR-128-3p, miR-376c-3p, miR-26a-5p, miR-191-5p) reached statistical significance. Of these, miR-191-5p had the strongest upregulation in PPMS (FC = 1.9). Analysis between the groups revealed significant upregulation of 5 miRNAs (miR-128-3p, miR-376c-3p, miR-26a-5p, miR-191-5p, miR-24-3p) in PPMS compared to HC. After the Bonferroni correction, 4 of them (miR-128-3p, miR-376c-3p, MIR-191-5p, miR-24-3p) remained statistically significant. miR-191-5p showed the greatest upregulation (FC = 2.3). In SPMS, 3 miRNAs (miR-376c-3p, miR-26a-5p, miR-191-5p) were upregulated (FC > 1.5, P < 0.05), but only miR-191-5p remained statistically significant after the Bonferroni correction (FC = 1.6). Comparison between PPMS and SPMS showed that 2 miRNAs (miR-128-3p, miR-24-3p) were significantly upregulated in PPMS after the Bonferroni correction (miR-128-3p, FC = 1.5; miR-24-3p, FC = 1.5). In the screening and validation phases, 18 MS and 9 HC samples were the same. To confirm that the results could be replicated in an independent cohort, additional logistic regression model analyses were performed excluding all the overlapping samples. Analysis showed that 3 miRNAs (miR-128-3p, miR-26a-5p, miR-191-5p) were significantly expressed in progressive MS patients (PPMS and SPMS) compared to HC as was detected in the whole validation cohort. The expression of miR-376c-3p did not differ statistically between MS and HC. Comparison between the groups showed that 3 miRNAs (miR-128-3p, miR-26a-5p, miR-191-5p) were significantly expressed in PPMS and 2 miRNAs (miR-26a-5p, miR-191-5p) in SPMS compared to HC. No differences in the expression levels were detected between PPMS and SPMS. However, the independent cohort was of very small size (n = 13) for PPMS vs. HC and for PPMS vs. SPMS. In PPMS vs. HC, expression of miR-376-3p was on border of statistical significance (P = 0.07) and for PPMS vs. SPMS, expression of miR-128-3p was on border of statistical significance (P = 0.07). In the whole MS group, expression of miR-124-3p correlated positively with time from first symptoms. The SPMS group had a positive correlation between expression of miR-128-3p and progression index. In the PPMS group, miR-124-3p correlated with time from first symptoms and miR-375 with age. When patients with EDSS > 6 were excluded from the analysis, a negative correlation was found between miR-375 and progression index in PPMS. | By ROC analysis, high values for AUC, sensitivity and specificity were obtained for miR-128-3p (AUC 0.727, sensitivity 0.645, specificity 0.714) and miR-191-5p (AUC 0.808, sensitivity 0.742, specificity 0.810) in PPMS, suggesting them as potential predictors of PPMS. | Increased expression of miR-191-5p was associated with PPMS and SPMS, while that of miR-128-3p was mostly associated with PPMS. | |||||||||
| Sharaf-Eldin et al., 2017; Egypt | 18 RRMS patients, 4 M/14 F, 29.1 ± 7.2 yrs, disease duration 5.5 yrs, EDSS 2.8, MSSS 4.6; 19 SPMS patients, 5 M/14 F, 37.7 ± 8.0 yrs, disease duration 8.0 yrs, EDSS 6.6, MSSS 8.4. Diagnoses of MS were based on the revised McDonald criteria (Polman et al., 2011). Disability and MS severity were evaluated according to EDSS and multiple sclerosis severity score (MSSS) (Roxburgh et al., 2005). None of the patients had received glucocorticosteroids, immunomodulators, immunosuppressants or other DMT for at least 3 weeks prior to study entry. Venous blood samples were collected, centrifuged (4500 r/min, 30 minutes) and sera stored at –80oC. | 23 HC, 6 M/17 F, 35.8 ± 9.8 yrs. They had no sign or history of autoimmune, inflammatory or neurological diseases. | By RT-qPCR, miR-145 and miR-223 (FC = 2.0 and FC = 2.2, respectively) were significantly upregulated in MS patients (n = 37) compared to HC, whereas miR-326 expression (FC = 1.8) did not differ significantly between MS patients and HC. For RRMS patients, FC values for miR-145, miR-223 and miR-326 were 2.6, 2.7 and 3.1, respectively, while for SPMS patients FC values for miR-145, miR-223 and miR-326 were 1.4, 2.2 and 1.2, respectively. FC value for miR-326 was significantly different between RRMS and SPMS. Expression of target genes SP1 (specificity protein 1) was significantly downregulated in MS patients compared to HC, whereas that of SMAD3 (signaling mother against decapentaplegic peptide 3) was not significantly downreguated in MS patients compared to HC. EDSS or MSSS did not correlate significantly with the expression levels of any of the miRNAs or mRNAs investigated. Regarding treatments, there were no significant differences in expression levels of miR-145, miR-223, miR-326, SP1, and SMAD3. | By ROC analysis, MS patients could be distinguished from HC by expression of miR-145 with AUC value 0.670 (sensitivity 0.730, specificity 0.609), expression of miR-223 with AUC value 0.702 (sensitivity 0.730, specificity 0.609. Combining miR-145 and miR-223 gave AUC value 0.713 (sensitivity 0.892, specificity 0.478). Also combining miR-145, miR-223 and miR-326 gave AUC value 0.725 (sensitivity 0.892, specificity 0.478). | Expression of miR-145 and miR-223 can distinguish MS patients from HC. | |||||||||
| Regev et al., 2017; USA | Cohort 1: 41 MS patients 29 RRMS/7 SPMS/5 PPMS, 7 M/34 F, 47.7 ± 9.5 yrs, disease duration from first symptoms 8.5 ± 7.6 yrs, EDSS 2.2 ± 2.5, DMT at MRI Untreated28/Glatiramer acetate4/beta interferons9. Cohort 2: 79 MS patients 66 RRMS/10 SPMS/3 PPMS, 26 M/53 F, 43.0 ± 7.5 yrs, disease duration from first symptoms 11.7 ± 6.8 yrs, EDSS 2.0 ± 1.9, DMT at MRI Untreated6/ Glatiramer acetate30/beta interferons 25/other 18. Serum samples were collected in tubes without additives, centrifuged (2000 × g, 10 minutes) to separate serum, and stored at –70oC. MRI scans were performed to identify brain lesions, brain atrophy, and cervical spinal cord lesions and atrophy. | MiRNAs were measured by RT-qPCR. In cohort 1, the miRNAs that demonstrated strong correlations with MRI were similar for brain parenchymal fraction and global cerebral GM fraction. However, the miRNAs associated with lesions were mostly different from those associated with atrophy, suggesting different pathogenic processes underlying focal lesions (inflammatory demyelination) vs. neurodegeneration (axonal and neuronal loss). In cohort 2, miRNAs that showed a strong correlation with whole-brain atrophy also indicated a strong correlation with cerebral GM atrophy. However, a different set of miRNAs correlated with spinal cord vs. brain atrophy. Although several mRNAs were associated with MRI outcome, none of these associations remained significant when correcting for multiple comparisons using false discovery rate. Several miRNAs showed significant associations with EDSS score in both cohorts. These associations for seven miRNAs miR-19a-3p, miR-101-3p, miR-30e-3p, miR-19b-3p, miR-29c-3p, miR-32-5p, miR-195-5p remained significant after correcting for multiple comparisons using false discovery rate. All of these miRNAs except for miR-195-5p had a protective relationship shown by a higher miRNA expression being associated with lower severity of MRI-indicated involvement. MiR-195-5p had a pathogenic relationship shown by a higher miRNA expression being associated with greater severity of MRI-indicated involvement. | Significant correlations were found between miRNAs and disability for miR-19a-3p, miR-101-3p, miR-30e-3p, miR-19b-3p, miR-29c-3p, miR-32-5p, miR-195-5p. Although several miRNAs were associated with MRI outcomes, none of these associations remained significant when correcting for multiple comparisons, suggesting that further validation of the findings is needed. | |||||||||||
| Regev et al., 2016; USA | Discovery phase: 7 RRMS patients, 2 M/5 F, 50 ± 6.4 yrs, disease duration 8.7± 1.5 yrs, EDSS 0.5 ± 0.8; 9 SPMS patients, 3 M/6 F, 50 ± 6.4 yrs, disease duration 17.0 ± 7.7 yrs, EDSS 5.8 ± 1.4; 10 PPMS patients, 5 M/5 F, 57 ± 7.5 yrs, disease duration 16.6 ± 5.2 yrs, EDSS 5.1 ± 2.3. Validation phase: 29 RRMS patients, 10 M/19 F, 36 ± 7.6 yrs, disease duration 3.4 ± 3.0 yrs, EDSS 1.1 ± 0.9; 19 SPMS patients, 6 M/13 F, 46 ± 6.9 yrs, disease duration 15.2 ± 4.8 yrs, EDSS 6.1 ± 1.2; 10 PPMS patients, 5 M/5 F, 47 ± 5.5 yrs, disease duration 10.9 ± 5.9 yrs, EDSS 5.3 ± 2.1. Samples from patients with MS were obtained from the CLIMB study. Those patients selected for this study had not received treatment with steroids in the past month; glatiramer acetate, interferon beta, fingolimod, dimethyl fumarate., or teriflunomide in the past 3 months; or other DMT in the past 6 months including cyclophosphamide, rituximab, daclizumab, methotretate, and natalizumab. Blood samples were collected in tubes without additives, and kept at room temperature for 30–60 minutes. Samples were centrifuged (2000 r/min, 10 minutes) to separate serum and then stored at –70oC. | Discovery phase: 20 HC, 6 M/14 F, 33 ± 9.2 yrs. Validation phase: 30 HC, 9 M/21 F, 43 ± 1.2 yrs. HCs were obtained from the Brigham PhenoGenetic Cohort study and from healthy participants enrolled in the CLIMB study. | By RT-PCR, in the discovery phase 167 miRNAs were identified that were differentially expressed between patients with MS and HC. After filtering using the selection criteria, 40 miRNAs were chosen for further validation. In the validation phase, 7 miRNAs were significantly differentially expressed in MS compared to HC. Of these 7 miRNAs, 6 were significantly different after correcting for multiple comparisons using false discovery rate: miR-320a, miR-486-5p, miR-320b, miR-25-3p, miR-140-3p (all upregulated) and let-7c-5p (downregulated). The findings remained unchanged after adjustment for age and gender. | By ROC analysis, the highest AUC value was for miR-320a (0.707) and discriminated MS patients from HC. When all 6 miRNAs that were significant after correcting for multiple comparisons were combined (miR-320a + miR-486-5p + miR-320b + miR-25-3p + miR-140-3p + let-7c-5p) the AUC value was 0.795. To identify disease category biomarkers, in the discovery phase 21 miRNAs were identified that were differentially expressed between the groups. In the validation phase, miR-27a-3p and miR-376-3p were significantly differentially expressed in RRMS compared to SPMS in the same direction in the discovery and validation phases. MiR-27a-3p had the highest AUC value (0.78) and only this miRNA remained significant after correcting for multiple comparisons. The results were the same after adjustment for age and gender. In the comparison of RRMS to PPMS, none of the miRNAs selected from the discovery phase were validated in the validation phase. To investigate the association between miRNAs and disability, in the discovery phase using all untreated patients with MS (plus 59 additional patients with RRMS), 103 miRNAs were significantly associated with EDSS score, and 40 were selected for further validation. In the validation phase, 10 miRNAs were significantly associated with EDSS score, and 9 remained significantly correlated to EDSS after adjusting for age. Two of these miRNAs miR-199a-5p, and miR-142-5p remained significantly correlated to EDSS after correcting for multiple comparisons. The highest correlation with EDSS was with miR-199a-5p. | Serum miR-320a, miR-486-5p, miR-320b, miR-25-3p, miR-140-3p, let-7c-5p discriminated MS from HC. MiR-27a-3p distinguished RRMS from SPMS. Also miR-199a-5p, and miR-142-5p were significantly correlated to EDSS score. | |||||||||
| Exosomes isolated from serum | ||||||||||||||
| Selmaj et al., 2017; Poland | Discovery set: 9 RRMS relapse patients, 1 M/8 F, 39 ± 11 yrs, disease duration 6.8 ± 7.8 yrs, EDSS 2.6 ± 1.9; 10 RRMS remission patients, 2 M/8 F, 37 ± 10 yrs, disease duration 6.4 ± 5.5 yrs, EDSS 2.3 ± 1.2. Validation set: 33 RRMS relapse, 8 M/25 F, 36 ± 11 yrs, disease duration 6.9 ± 8.2 yrs, EDSS 2.6 ± 1.1; 30 RRMS remission, 9M/21F, 37 ± 9 yrs, disease duration 7.5 ± 8.8 yrs, EDSS 2.2 ± 1.3. MS patients were classified as RRMS according to the revised McDonald criteria. Relapse was defined as the appearance of new neurological signs or worsening of pre-existing signs after a minimum of 30 days of clinical stability. All patients were sampled before methylprednisolone administration. Patients in remission did not receive DMT for at least 6 months. Venous blood was collected, allowed to coagulate, centrifuged, and serum removed and stored at –80oC. All patients were assessed for EDSS and examined by MRI. Exosomes were isolated from serum samples by the polymer formulation method (Peterson M et al., 2015). The vesicles isolated from sera were confirmed to be exosomes based on size (30–100 nm) as determined by nanoparticle tracking analysis (NTA) and expression of exosomal protein markers CD9 and Alix. There were no significant differences in total number of exosomes in sera of RRMS patients in relapse and in remission or HC. There were no significant differences in total amount of exosome RNA in sera of RRMS patients in relapse or remission and HC | Discovery set: 10 HC, 1 M/9 F, 34 ± 10 yrs. Validation set: 32 HC, 8 M/24 F, 38 ± 10 yrs. | MiRNA profiling of exosomes by next generation sequencing (NGS) in 29 samples of the discovery set identified 4 miRNAs that were significantly differentially expressed in RRMS patients in remission and relapse and HC (miR-122-5p, miR-196b-5p, miR-301a-3p, miR-532-5p). The specificity of these 4 serum exosomal miRNAs for RRMS patients was validated in a separate cohort of 95 subjects by qPCR. All 4 miRNAs were significantly differentially expressed between RRMS in remission and relapse and HC. In agreement with the NGS data, miR-122-5p, miR-196b-5p, miR-532-5p were significantly downregulated in RRMS relapse patients when compared to RRMS remission patients. All 4 miRNAs were significantly downregulated in RRMS relapse compared to HC, and miR-122-5p was significantly downregulated in RRMS remission compared to HC. | By ROC analysis, these 4 miRNAs gave high AUC values for discriminating between RRMS patients in relapse and HC (for miR-122-5p, miR-196b-5p, miR-301a-3p, miR-532-5p the values were 0.878, 0.866, 0.681, 0.800, respectively). Combining the levels of miR-122-5p and miR-196b-5p provided the highest AUC value for discriminating RRMS relapse from HC (0.904) and for RRMS relapse from RRMS remission (0.866). A combined measurement of miR-122-5p and miR-196b-5p improved diagnosis of RRMS relapse vs. remission with sensitivity > 0.89 and specificity > 0.75. No correlation was found between the clinical parameters (disease duration and disability assessed by EDSS) and the levels of the 4 miRNAs tested. To determine whether the levels of the 4 miRNAs correlated with MRI evidence of disease activity, the RRMS samples were divided into 2 categories: patients with Gd-enhancing lesions on brain MRI (Gd- positive) and patients with no signs of Gd enhancement (Gd- negative). The Gd- positive patients had significantly lower serum concentrations of all these miRNAs. Thus, lower levels of circulating exosomal miR-122-5p, miR-196b-5p, miR-301a-3p, miR-532-5p were related to disease activity. | Exosomal miRNAs might be a useful biomarker to distinguish MS relapse. PBMC secretion of all 4 miRNAs was significantly downregulated in RRMS patients compared to HC. PBMCs might represent a potent source of the exosomes containing miR-122-5p, miR-196b-5p, miR-301a-3p, and miR-532-5p, the secretion of which are decreased in RRMS patients. | |||||||||
| Niwald et al., 2017; Poland | 23 stable RRMS patients, 8 M/15 F, 48.8 ± 11.2, disease duration 13.2 ± 7.2 yrs, EDSS 5.5 ± 2.1, MoCA (Montreal cognitive assessment index) 25.9 ± 2.7, BDI (Beck Depression Index) 9.5 ± 5.3; 13 postacute RRMS patients, 3 M/10 F, 41.8 ± 10.2, disease duration 9.8 ± 9.6 yrs, EDSS 6.3 ± 2.8, MoCA 25.2 ± 4.1, BDI 11.7 ± 3.3. The stable RRMS patients were in the remission phase, without treatment, for over 2 yrs without attacks or steroid treatment (late phase). The postacute RRMS patients were in the early phase of remission, 1 to 2 mths free of i.v. 5-day methyl-prednisolone (1000 mg/d) due to severe exacerbation in Neurological units. All the patients underwent complete medical examinations. Venous blood was collected into tubes containing an anticoagulant (EDTA) and left at room temperature until clot formation (30–60 minutes). Blood samples were centrifuged (1200 × g, 10 minutes, 23oC) and the serum carefully removed and stored at –80oC. Exosome isolation from frozen human serum was performed. Serum samples were thawed, and centrifuged (2000 × g, 30 minutes) to remove the cells and residues from the serum. Then, 500 µL of the supernatant was treated with 0.2 volumes of the Total Exosome Isolation reagent and after mixing well were incubated 2–8oC for 30 minutes. After incubation, samples were centrifuged (10,000 × g, 10 minutes, room temperature). The supernatant was removed and the pellet containing the exosomes was suspended in 200 µL PBS and stored at 2–8oC for 1 week or at –20oC until RNA isolation. | 10 HC, 3 M/7 F, 50.7 ± 8.1 yrs. | RNA isolation from the exosomes was performed. Analysis by qPCR showed a decreased expression of miR-155 and miR-301a (in 94% and 51% samples, respectively) and an increased expression of miR-326 (in 72% samples) in RRMS patients (n = 36). The difference in relative expression of miR-155 for RRMS patients compared to HC was statistically significant. No correlation was found between expression levels of miR-155, miR-326 and miR-301a and age. A positive correlation was found between relative expression of miR-326 and miR-301a and BDI. A positive correlation was found between RQ (relative quantification) miR-301a and MoCA. Expression levels of miR-301a and miR-155 were higher in RRMS patients in postacute vs. stable phase of remission. Statistically significant differences in RQ values between early vs. late phase of remission were found for miR-301a and miR-155. There was no significant difference in the case of miR-326. Statistical analysis between two groups of RRMS patients with BDI ≤ 11 vs. BDI > 11 revealed lower miR-326 and miR-301a expression levels in patients with BDI ≤ 11, while miR-155 expression was increased in BDI ≤ 11. A statistically significant difference was found for miR-326, but not for miR-301a and miR-155. Statistical analysis between HC, RRMS BDI > 11 and RRMS BDI ≤ 11 showed significant differences only for miR-326, with a significantly higher expression in RRMS BDI > 11 vs. HC. On dividing RRMS patients into two subgroups based on their disability EDSS ≤ 5 and EDSS > 5, no significant differences were found in expression level of miR-155, miR-326 and miR-301a. | The expression levels of miR-301a and miR-155 were significantly higher in the postacute vs. stable phase of remission, where neuroinflammatory processes are more severe. Expression changes of serum miRNAs could be used as a biomarker of relapse activity. | ||||||||||
| Ebrahimkhani et al., 2017; Australia | 14 RRMS patients, 4 M/10 F, age of onset 35.6 ± 7.3 yrs, disease duration 6.9 ± 7.1 yrs, treatment Y 6/N 8, EDSS 1.5 ± 1.0. Discovery: 11 SPMS7/PPMS4 patients, 6M/5F, 38.4 ± 8.5 yrs, disease duration 15 ± 9.4 yrs, treatment Y 4/N 7, EDSS 5.3± 1.6. Validation: 11 SPMS/PPMS patients, 1 M/10 F, 32.3 ± 8.2 yrs, disease duration 20.4 ± 4.8 yrs, treatment Y 7/N 4, EDSS 6.0 ± 1.1. MS was diagnosed according to the revised McDonald criteria. Venous blood was collected at the time of clinical consultation, left at room temperature for 30 min for coagulation, and then centrifuged (1800 × g, 10 minutes). The resulting serum was centrifuged (3000 × g, 20 minutes) to remove any cellular debris, and stored at –80oC. Serum samples were treated with RNaseA to remove any unprotected circulating RNA. Exosomes were isolated from 1 ml of serum by size exclusion chromatography. Nanoparticle tracking analysis and transmission electron microscopy revealed a population of nanovesicles with a predominant size of 95 nm and cup-shaped morphology typical of exosomes. Western blotting of protein extracts for CD61, CD83 and Alix confirmed that the particles isolated expressed all three characteristic exosome markers. | 11 HC, 2 M/9 F, 40.3 ± 13.3 yrs, treatment Y 0/N 11. | Using NGS and three statistical approaches, 4 significantly upregulated miRNAs were identified for RRMS patients compared to HC (miR-15b-5p, miR-30b-5p, miR-342-3p, miR-451a), and 4 significantly upregulated miRNAs for SPMS/PPMS patients compared to HC (miR-127-3p, miR-370-3p, miR-409-3p, miR-432-5p). There were 3 significantly upregulated miRNAs for RRMS compared to SPMS/PPMS patients. MiRNAs were identified as differentially expressed if they met a fold-change ≥ 2 and P ≤ 0.05 in at least two of the three statistical tests. In the validation study, 6 miRNAs were significantly upregulated in SPMS/PPMS patients compared to HC (miR-370-3p, miR-409-3p, miR-432-5p, miR-15b-5p, miR-223-3p, miR-23a-3p). In addition, 9 miRNAs were dysregulated in RRMS compared to SPMS/PPMS patients (miR-15b-5p, miR-23a-3p, miR-223-3p, miR-30b-5p, miR-342-3p, miR-374a-5p all upregulated, and miR-432-5p, miR-433-3p, miR-485-3p all downregulated (P < 0.05 in at least two tests and FC ≥ 1.7 in either direction)). | By ROC analysis, miR-451a had the highest AUC value (0.83) for distinguishing RRMS patients from HC, while miR-127-3p, miR-409-3p, miR-370-3p, miR-432-5p had AUC values 0.90, 0.91, 0.86, 0.90, respectively, for distinguishing SPMS/PPMS patients from HC. For distinguishing RRMS from SPMS/PPMS patients, miR-433-3p, miR-432-5p, miR-485-5p had AUC values 0.93, 0.86, 0.87, respectively. Using the original 9 miRNAs identified for RRMS vs. SPMS/PPMS it was possible to predict 11/11 PPMS samples in the validation set. | Differentially expressed exosomal miRNAs were identified in both RRMS (miR-15b-5p, miR-451a, miR-30b-5p, miR-342-3p) and progressive MS patient sera (miR-127-3p, miR-370-3p, miR-409-3p, miR-432-5p) compared to HC controls. A group of nine miRNAs (miR- 15b-5p, miR-23a-3p, miR-223-3p, miR-374a-5p, miR-30b-5p, miR-433-3p, miR-485-3p, miR-342-3p, miR-432-5p) were found that distinguished RRMS from progressive disease. | |||||||||
AUC: Area under curve; CI: cognitively impaired; CP: cognitively preserved; DMT: disease-modifying treatment; EDSS: expanded disability status scale; F: females; FC: fold change; M: male(s); MS: multiple sclerosis; N: no; NA: not available; PPMS: primary-progressive MS; ROC: receiver operating characteristics; RRMS: relapsing-remitting MS; SPMS: secondary-progressive MS; Y: yes.