Effect of miR-21 in mesenchymal stem cells-derived extracellular vesicles behavior

Abstract

Background: A challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation.

Methods: Mesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP.

Results: Syndecan-1 (SDC1) was the most decreased protein in MSC-miR21^--derived EV, and it was involved in inflammation and EV production. MSC-miR21^--derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21⁺-derived EV.

Conclusion: This work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1.

Keywords: Extracellular vesicles (EV); Inflammaging; Mesenchymal stem cells (MSC); Senescence-associated secretory phenotype (SASP); Syndecan-1 (SDC1); miR-21-5p (miR-21).

Fig. 1

Characterization of MSCs from umbilical cord stroma and their EV. A One representative fluorescence-activated cell sorting (FACS) assay is shown. Positive MSC markers (CD105, CD90 and CD73) and negative hematopoietic markers (CD34 and CD45). B Representative pictures of immunohistochemical analysis (OR = Oil Red O, SafO = safranin O and AR = Alizarin Red from human umbilical cord stroma after 14 days with specific differentiation medium (DMEM, AD = Adipocyte medium, CH = Chondrocyte medium and OS = Osteocyte medium). C Histogram represents gene expression of pluripotency markers, Nanog, Sox9 and Oct4 in MSC in front of TC28a2 cell line. Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of HPRT gene used as housekeeping. *P value less than 0.05 was considered statistically significant using two-way ANOVA test (n = 3). D Representative result from the NTA assay of MSC-derived EV. E Electron micrograph of MSC-derived EV (scale bar = 200 nm). F Immunoblot staining for exosome markers CD63 and Syntenin-1, Calnexin and GM130 as a negative control. Full-length blots/gels are presented in Additional file 3: Fig. S3

Fig. 2

Effect of inhibition of miR21 and MSC-miR-21⁻-derived EV. A Histogram represents gene expression of SASP and inflammaging markers (S100A4, S100A6, HMGB1, IL-6, TLR4 and IL-1β) from MSC-mimic and MSC-miR-21⁻. Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of HPRT gene used as housekeeping. B Representative result from the NTA assay of MSC-mimic-derived EV. C Representative result from the NTA assay of and MSC-miR-21⁻-derived EV. D Histogram represents gene expression of miR-21 in MSC-mimic and MSC-miR-21⁻. Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of U6 gene used as housekeeping. E Histogram represents number of particles in MSC-mimic and MSC-miR21⁻ by NTA. F Histogram represents size of particles in MSC-mimic and MSC-miR21⁻ by NTA. Bars means ± SEM from three independent experiments.*P value less than 0.05 was considered statistically significant two-way ANOVA test (n = 3)

Fig. 3

Shotgun study of miR21 in protein cargo of MSC-derived EV. A Workflow Peptides from MSC-mimic-derived EV and MSC-miR-21⁻-derived EV with 2 or 3 biological replicates were labeled with 7 TMT reporter from 10-plex reagents. These reagents are observed as a monoisotopic complex in the first round of MS analysis on a high-resolution mass spectrometer. During MS/MS and HCD-based fragmentation, the TMT labels peptides are fragmented to produce 8 reporter ions with distinguishable masses in the low m/z range, which allow relative protein quantitation based in their intensities ratios. B Volcano plot of all proteins identified in our shotgun study for the MSC-mimic and MSC-miR21⁻. SDC1 is the differentially expressed protein that was significantly downregulated by inhibition of miR-21 (fold change ≥ 2; P value ≤ 0.05). C Orthogonal validation of SDC1 in MSC-miR-21⁻ (miR-21.⁻), MSC-mimic (mimic) by western blot. B-actin was used as housekeeping. Full-length blots/gels are presented in Additional file 3: Fig. S3

Fig. 4

Paracrine effect of MSC-miR21⁻ -derived EV vs MSC-miR21⁺-derived EV in vitro. A Workflow of experiment using MSC-miR-21⁻-derived EV or MSC-miR-21⁺-derived EV to treat MSC-miR-21⁻ for 3 days. After that the cells were collected to performed the successive experiments. B Histogram represents gene expression of miR-21 in MSC-mimic and MSC-miR-21⁺ transitory transfected. Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of U6 gene used as housekeeping. *P value less than 0.05 was considered statistically significant. C Histogram represents gene expression of SASP and inflammaging markers (S100A4, S100A6, HMGB1, IL-6, TLR4 and IL-1β) from MSC-mimic and MSC-miR-21⁻ treated with MSC-miR-21⁻-derived EV. D Western blots analysis of MAPK family (ERK1/2, AKT, phosphor-ERK1/2, phosphor-AKT) and SDC1 from MSC-miR-21⁻ lysates described in the experiment above (A). Full-length blots/gels are presented in Additional file 3: Fig. S3. E Histogram represents gene expression of SASP and inflammaging markers (S100A4, S100A6, HMGB1, IL-6, TLR4 and IL-1β) from MSC-mimic and MSC-miR-21⁻ treated with MSC-miR-21⁺-derived EV. Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of HPRT gene used as housekeeping. *P value less than 0.05 was considered statistically significant. F Densitometry results for normalized proteins relative to GAPDH. Bars are means ± SEM from three independent experiments. *P < 0.05 was considered statistically significant using two-way ANOVA test (n = 3)

Fig. 5

Validation of SDC1 in vitro. A Workflow of experiment using MSC-miR-21⁺ treated with SSTN during 3 days. After that the cells and their derived-EV were collected to performed the successive experiments. B Histogram represents gene expression of miR-21 in cells collected from workflow experiment described in (A). Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of U6 gene used as housekeeping. C Histogram represents gene expression of SASP and inflammaging markers (S100A4, S100A6, HMGB1, IL-6, TLR4, IL-1β) from cells collected from workflow experiment described in (A). Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of HPRT gene used as housekeeping. D Histogram represents number of particles from cells collected from workflow experiment described in (A). Bars are means ± SEM from three independent experiments. *P value less than 0.05 was considered statistically significant using two-way ANOVA test (n = 3)

Fig. 6

Paracrine validation of SDC1 in vitro. A Workflow of experiment using MSC treated with MSC-miR-21⁺-derived EV or MSC-miR-21⁺-derived EV previously treated with SSTN, for 3 days. After that the cells were collected to perform the successive experiments. B Histogram represents gene expression of miR-21 in cells collected from workflow experiment described in (A). Real-time reverse transcriptase PCR (RT-qPCR) analysis normalized by expression of U6 gene used as housekeeping. C Histogram represents gene expression of SASP and inflammaging markers (S100A4, S100A6, HMGB1, IL-6, TLR4 and IL-1β) from cells collected from workflow experiment described in (A). D Histogram represents number of particles from cells collected from workflow experiment described in (A). Bars are means ± SEM from three independent experiments. *P value less than 0.05 was considered statistically significant using two-way ANOVA test (n = 3)