Hsa-miR-21-5p is induced by interleukin-6 and affects multiple pathogenic factors associated with fibroblast-like synoviocytes in rheumatoid arthritis

Fig. 1. The intronic type is a large population of microRNAs expressed in FLS. (a) Schematics of intergenic and intronic miRNAs. (b,c) Small RNA-seq performed for 2861 mature miRNAs in fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA) (n = 2, from each individual subject). 297 species of mature miRNAs were detected. (b) The distribution ratio for the types of miRNAs. 297 miRNAs were categorized by their respective types (e.g., intergenic, intronic, exonic, and an unclassifiable group). (c) Top 20 most highly expressed intronic miRNAs, displayed with read count of small RNA-seq.

Fig. 2. Alteration of intronic microRNA expression levels in cytokine-stimulated FLS. The FLS data were collected from patients with rheumatoid arthritis (RA). FLS were stimulated with IL-6 (100 ng/mL, ■) with 100 ng/mL of sIL-6R, TNFα (100 ng/mL, ▲), IL-1β (10 ng/mL, ▼), and IL-17 A (100 ng/mL, ◆) for 24 h. (a) hsa-miR-191-5p expression. (B) hsa-miR-148b-3p expression. (c) hsa-miR-93-5p expression. (d) hsa-miR-140-3p expression. (e) hsa-miR-423-5p expression. (f) hsa-miR-27b-3p expression. (g) hsa-miR-15b-5p expression. (h) hsa-miR-342-3p expression. (i) hsa-miR-21-3p expression (j) hsa-miR-21-5p expression. All expression levels were measured using RT-qPCR, and statistical analyses were performed using Dunn’s test (*p < 0.05). Data represent the mean ± the standard error of the mean (n = 3, from each individual subject).

Fig. 3. Pri-miR-21 in FLS is induced by IL-6 through STAT3, independent of host VMP1 expression. (a) The schema of the VMP1 gene locus on chromosome 17 in Homo sapiens. Red arrow indicates the location of PCR primers for Exon 5, Exon 12, pre-miR-21, and pri-miR-21-3’UTR. (b–d) Expression levels were measured using RT-qPCR. (b) Whole synovial tissues collected from patients with rheumatoid arthritis (RA; n = 10) and osteoarthritis (OA; n = 6). Data represent the mean ± the standard deviation, and statistical analysis was performed using U-test. (c, d) Pri-miR-21-3’UTR expression levels in fibroblast-like synoviocytes (FLS) collected from patients with RA. Data represents the mean ± the standard error of the mean, and statistical analysis was performed using Dunn test. (c) Time-dependent stimulation with recombinant IL-6 (100 ng/mL) and recombinant sIL-6R (100 ng/mL) (n = 3). (d) Stimulation with IL-6 (100 ng/mL) and sIL-6Rα (100 ng/mL) for 24 h, and small interfering RNA against STAT3 for 34 h (n = 6). VMP1, vacuole membrane protein 1.

Fig. 4. RNA-seq analysis of hsa-miR-21-5p-induced gene expression changes in FLS. RNA from miR-21-5p or miR control (miRNA negative control) mimic-transfected FLS was collected and analyzed using next-generation sequencing analysis (conventional RNA-seq) (n = 2, from each individual subject). (a) Volcano plot, analyzed with TPM method. (b) Volcano plot, analyzed with FPKM method. (c) Represents the top five downregulated genes. (d) The Venn diagram illustrates the relationship between downregulated genes detected with conventional RNA-seq analysis and genes that have potential target sequences for hsa-miR-21-5p, as predicted by the TargetScan database. DDAH1, dimethylarginine dimethylaminohydrolase 1; TNFRSF11B, TNF receptor superfamily member 11b; OPG, osteoprotegerin; RP2, retinitis pigmentosa 2; ID3, inhibitor of DNA binding 3; PDCD4, programmed cell death 4.

Fig. 5. Hsa-miR-21-5p promotes fibroblast-like synoviocytes proliferation. (a, b) MH7A cells were transfected with miR-21-5p mimic or miR control. (a) PDCD4 expression levels were measured using RT-qPCR (n = 3). (b) PDCD4 proteins were detected by Western blotting. (c) Immunohistochemistry (IHC) staining for PDCD4 in formalin-fixed paraffin-embedded (FFPE) synovial tissues which were harvested from patients with RA and OA (red, PDCD4; purple, hematoxylin). Black scale bar = 100 μm. (d) Cell viability assay using calcein-AM for MH7A cells transfected with miR-21-5p mimic or miR control (n = 3). (e, f) MH7A cells were transfected with PDCD4 IVT mRNA or mock IVT mRNA. (e) Cell viability assay using calcein-AM for MH7A cells at 24 h after transfection (n = 3). (f) PDCD4 proteins were assayed with Western blotting. (a, d, e) Data are presented as mean ± the standard error of the mean, t test. PDCD4, programmed cell death 4.

Fig. 6. Hsa-miR-21-5p might indirectly influence synovial functions. (a, b, e, f) MH7A cells were transfected with miR-21-5p mimic or miR control. (c, g) Primary cultured FLS were transfected with miR-21-5p mimic or miR control. (a) RT-qPCR for SEMA5A expressions (n = 3). (b, c) SEMA5A proteins were detected by Western blotting. (d, h) Whole synovial tissues collected from patients with RA and OA. (d) RT-qPCR for SEMA5A expressions (RA; n = 11 vs. OA; n = 9). (e) RT-qPCR for OPG expressions (n = 3). (f, g) Supernatants of each cell after transfection were collected. Cytometric bead array (CBA) assay was performed to measure the OPG protein concentrations (n = 3). (h) RT-qPCR for OPG expressions (RA; n = 11 vs. OA; n = 9). (a, e, f, g) Data are presented as mean ± the standard error of the mean, t test. (d, h) Bars represent the mean value, and statistical analysis was performed using U-test. (i) The schema shows the latent function of hsa-miR-21-5p. MiR-21-5p decreases PDCD4 expression, thereby enhancing cell proliferation. It could also upregulate SEMA5A, which contributes to inflammation and enhances osteoclastogenesis through the reduction of OPG. SEMA5A, semaphorin 5A; OPG,…