Fig 1.
MiR-200c-3p and stemness markers expression during in vitro passages in ASCs.
A. MiR-200c-3p expression by qRT-PCR, during in vitro passages (P). MiR-200c-3p expression was normalized to U6. SD± are the mean of three independent experiments. Dunnett’s multiple comparisons test was applied to compare P2 with each of the passages. *p<0.05, **p<0.01, ***p<0.001. B. Stemness markers during the in vitro passages were normalized to GAPDH. SD± are the mean of three independent experiments. Ordinary one-way Anova, Dunnett’s multiple comparisons test was performed for each stemness marker and p values were calculated comparing P2 with the rest of the passages. *p<0.05, **p<0.01, ****p<0.0001.
Fig 2.
Expression of proliferation/senescence markers at P4 and P9 passages in ASCs.
A. Expression analysis of miR-200c-3p at P4 versus P9 was performed by qRT-PCR and calculated as a fold change using 2-ΔΔCt. SD± is the mean of three experiments and the p values were calculated with two-tailed unpaired t test, *p<0.05, ***p<0.001. B. Western blot analysis of the proliferation-related markers p-ERK, ERK, cyclin D1, and the senescence-related markers p53 and p21, at P4 and P9. β-Actin was used as loading control. WBs were repeated twice. Densitometric analysis was performed with ImageJ Software (v.10.2) for each protein. Normalization was calculated as a ratio between the number of pixels (n.p.) of p-ERK divided to the n.p. of ERK and the rest of the proteins normalized with β-actin. SD± is the mean of two WBs and the p values were calculated with two-tailed unpaired t test, *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
MiR-200c-3p regulates the expression of stemness markers.
A. qRT-PCR of miR-200c-3p (i), Nanog (ii), Oct4 (iii) and Sox2 (iv) in ASCs at P4 transfected with miR-200c-3p mimic. The fold change of miR-200c-3p was normalized to U6 and that of stemness markers to GAPDH. SD± is the mean of three experiments and the p values were calculated with unpaired t test, *p<0.05, ***p<0.001. B. qRT-PCR of miR-200c-3p (i) in ASCs transfected with anti-miR-200c-3p. Nanog (ii), Oct4 (iii) and Sox2 (iv) expressions were normalized to GAPDH. SD± is the mean of three experiments and the p values were calculated with unpaired t test, **p<0.01. C. CD44 mRNA was detected by qRT-PCR (i) and protein expression of CD44, p-STAT3 and STAT3 was detected by WB (ii), in mimic-miR-200c-3p transfected ASCs. D. CD44 mRNA was detected by qRT-PCR (i) and protein expression of CD44, p-STAT3 and STAT3 was detected by WB (ii), in anti-miR-200c-3p transfected ASCs. GAPDH was used as housekeeping for qRT-PCR and β-actin in WBs, as a loading control. SD± is the mean of three experiments and the p values were calculated with unpaired t test, *p<0.05, **p<0.01.
Fig 4.
MiR-200c-3p affects proliferation and regulates senescence through p53, p21 expression in ASCs.
A. Representative images of ASCs transfected with mimic at P4, (low miR-200c-3p) (i) or inhibitor miR-200c-3p at P9, (high miR-200c-3p) (ii) and their corresponding controls. The images were taken at 72 h post-transfection at 40x magnification. B. Clonogenic assays of ASCs transfected with miR-200c-3p mimics (i) and anti-miR-200c (ii) or their respective controls. Cells were stained with crystal violet and subsequently photographed. Absorbance at 595 nm was measured to estimate the optical density in each condition. The black arrows indicate colonies formation. SD± is the mean of optical density of each condition. The experiment was repeated twice and each condition in triplicates and p values were calculated with unpaired t test, ***p<0.001. C. Expression of p-ERK, ERK, Cyclin D1 was studied by WB in ASCs transfected either with mimic at P4 or with anti-miR-200c-3p at P9. On the right, densitometry analysis measures the levels of p-ERK normalized to ERK and Cyclin D1 normalized to β-actin. SD± is the mean of two WBs and the p values were calculated with unpaired t test, **p<0.01. D. (i): The percentage of cells positive for SA-β-galactosidase in ASCs transfected with miRNA mimics or (ii): inhibitors was calculated by counting blue cells in at least 7–8 different areas and in triplicates for each condition. The images (magnification 20x) are representative of one out of three wells. The black arrows indicate β-gal positive cells. SD± is the mean of the % β-gal positive cells detected in three wells for each condition and the p values were calculated with unpaired t test, *p<0.05, **p<0.01. E. Detection of p53, p21 transcripts was performed by qRT-PCR in ASCs transiently transfected at 72 h with (i): mimic-miR-200c-3p, at P4, and the corresponding WBs or (ii): anti-miR-200c-3p, at P9 and the corresponding WBs. GAPDH was used for gene normalization in qRT-PCR and β-actin, loading control for proteins. SD± is the mean of three independent experiments. P values were calculated with two tailed unpaired t test, *p<0.05, ***p<0.001.
Fig 5.
MiR-200c-3p inverse correlates with PD-L1 expression and modulates PD-L1 and IDO1 expression in ASCs.
A. PD-L1 expression at P2-P15 passages was assessed by WB. Densitometric analysis measures the fold change of PD-L1 expression normalized to β-actin in each passage. One out of two WBs is shown. Dunnett’s multiple comparisons test was applied to compare P2 with each of the passages. *p<0.05, ***p<0.001. B. (i): PD-L1 mRNA detection by QRT-PCR, during in vitro passages (P). PD-L1 expression was normalized to GAPDH. SD± are the mean of three independent experiments. Dunnett’s multiple comparisons test was applied to compare P2 with each of the passages. *p<0.05, **p<0.01, ***p<0.001. (ii): Correlation analysis between PD-L1 and miR-200c-3p expression was performed in ASCs derived from 5 healthy donors at all passages from 2 to 15. The square of the correlation coefficient, R2 <0.7 shows that the inverse correlation is significant with p<0.01. C. (i): PD-L1 transcript fold change by qRT-PCR in ASCs transfected with miR-200c-3p mimics, at P4 and (ii): PD-L1 protein detection by WB. (iii): IDO1 transcript fold change by qRT-PCR in ASCs transfected with miR-200c-3p mimics, at P4. D. (i): PD-L1 transcript by qRT-PCR in ASCs transfected with miR-200c-3p inhibitors, at P9 and (ii): PD-L1 protein detection by WB. and (iii): IDO1 transcript fold change by qRT-PCR in ASCs transfected with miR-200c-3p inhibitor, at P9. SD± is the mean of three experiments and the p values were calculated with two tailed unpaired t test. **p<0.01, ***p<0.001.
Fig 6.
Graphical summary of how miR-200c-3p may regulate self-renewal, proliferation, senescence and have immunomodulatory effect in ASCs.
Left: in the cytoplasm, over-expression of miR-200c-3p (green arrow) may stimulate cell proliferation through an increase of p-ERK and inhibit cell senescence by downregulating p53/p21 axis (red arrow). MiR-200c-3p can also increase CD44 expression leading to STAT3 phosphorylation, which in turn may activate transcription of stemness-related genes in the nucleus. Right: Inhibition of miR-200c-3p with a specific anti-miR (red arrow) alleviates repression of PD-L1 and IDO-1, the inhibitory checkpoints, to regulate immune responses against ASCs.