Fig 1.
Flow cytometry of FoxP3+ CD4+ T cells and RORɣt+ CD4+ T cells.
CD4+ T cells were isolated by CD4+ Tcell isolation kit II human of Miltenyi Biotec and stained with respective antibodies and evaluated in relapsing phase (n = 20) and remitting phase (n = 20) of MS patients and healthy controls (n = 10). A forward and side scatter gate was used to select lymphocyte population and fluorescence compensation was set according to labeled lymphocytes with only green and only red fluorescent separately versus isotype control (A). Percentage of RORγt+ CD4+ T cells measured by Flow cytometry, shows meaningful increase in relapsing group (B) while percentage of FoxP3+ CD4+ T cells elevates in remitting group (C) (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric Mann-Whitney t-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).
Fig 2.
Up-regulation of miR-141 and miR-200a in relapsing phase of MS patients.
RT-qPCR analysis of miR-141 expression level in CD4+ T cells of MS patients in relapsing phase (n = 20), remitting phase (n = 20) and healthy controls (n = 10) (A). RT-qPCR analysis of miR-200a expression level in CD4+ T cells of MS patients in the same groups (B). Results are normalized relative to expression level of reference gene, RNU48 (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric Mann-Whitney t-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).
Fig 3.
The expression level of master markers of Th17& Treg cells and IL-17A in CD4+ T cells of relapsing group (n = 20), remitting group (n = 20) and healthy controls (n = 10) in MS patients.
Results are normalized relative to expression level of reference gene, 18srRNA (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric Mann-Whitney t-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).
Fig 4.
Heatmap view of pathways in which validated targets of miR-141 and miR-200a are involved.
The heatmap was drawn in consequent of enrichment analysis on miR-141 and miR-200a targetome (for valid targets) in which a merged p-value is calculated for each pathway by applying Fisher’s meta-analysis manner. The resulting p-value represents the examined pathways that are significantly enriched with gene targets of miR-141 and miR-200a. Color gradient displays the importance of mentioned pathways. Heatmap was drawn based on validated targets of miR-141 and miR-200a, according to DIANA miRPath. Notably, mTOR signaling pathway was specified as one of the major pathways based on its involvement in differentiation of Th17 cells. Analysis shows that miR-141 affects mTOR pathway more effective than miR-200a does.
Table 1.
Top statistically related signaling pathways to miR-141 and miR-200a targetome obtained from different databases by means of DAVID tool.
Fig 5.
miR-141 and miR-200a targetome are both involved in several important pathways including TGF-β, mTOR and FOXO, which their partial diagram collected from KEGG pathway is demonstrated.
Red stars mark common targets of miR-141 and miR-200a.
Fig 6.
Target genes of miR-141 and miR-200a specified in-silico were tested for further analysis and the expression level of target genes of miR-141 and miR-200a in CD4+ T cells of relapsing group (n = 20), remitting group (n = 20) and healthy controls (n = 10) were assessed.
Results are normalized relative to expression level of reference gene, 18srRNA (*p < 0.05 and **p < 0.01, non-parametric Mann-Whitney t-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).