Fig. 5
The miR-21/SP1/DNMT1 positive feedback loop in MSCs promoted miR-21 expression
(A)DNMT1 expression in human BM-MSCs and GA-MSCs was measured using mRNA sequencing. (B) DNMT1 expression in human BM-MSCs and GA-MSCs was measured using western blotting. Quantification of the fold change in the DNMT1/β-actin ratio (normalized to BM-MSCs) is shown. (C) miR-21 expression was measured in control and DNMT1 knockdown human BM-MSCs. (D) miR-21 expression was measured in control and DNMT1 knockdown human BM-MSCs-derived exosomes. (E) miR-21 expression was measured in BM-MSCs treated with the DNA methylation inhibitor 5-aza-2’-deoxycytidine. (F) MSP analysis was performed to examine the methylation status of CpG islands in the promoter region of miR-21 in BM-MSCs and GA-MSCs. (G) DNMT1 expression in human BM-MSCs transfected with miR-21 mimics was measured using western blotting. Quantification of the fold change in the DNMT1/β-actin ratio (normalized to NC) is shown. (H) BM-MSCs were cotransfected with miR-21 and a luciferase reporter containing the 3’UTR of DNMT1. The luciferase activity was measured. (I) The downregulated genes in GA-MSCs were filtered by overlapping them with predicted miR-21 targets (determined with starBase) and predicted DNMT1 transcription factors (determined with the PROMO database). (J) DNMT1 expression was measured in control and SP1 knockdown human BM-MSCs. Quantification of the fold change in the DNMT1/β-actin ratio (normalized to NC) is shown. (K) SP1 expression was measured in control and miR-21-overexpressing human BM-MSCs. Quantification of the fold change in the SP1/β-actin ratio (normalized to miR-NC) is shown. (L) Construction of wild-type (WT) and mutant-type (MUT) luciferase reporter vectors based on the predicted binding site of miR-21 in SP1.(M) The expression of DNMT1 in MSCs transfected with miR-NC or miR-21 mimics and nonsense sequence or SP1 overexpression plasmids was measured using western blotting. The data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001