Enhancer RNA commits osteogenesis via microRNA-3129 expression in human bone marrow-derived mesenchymal stem cells

Abstract

Background: Highly regulated gene expression program underlies osteogenesis of mesenchymal stem cells (MSCs), but the regulators in the program are not entirely identified. As enhancer RNAs (eRNAs) have recently emerged as a key regulator in gene expression, we assume a commitment of an eRNA in osteogenesis.

Methods: We performed in silico analysis to identify potential osteogenic microRNA (miRNA) gene predicted to be regulated by super-enhancers (SEs). SE inhibitor treatment and eRNA knocking-down were used to confirm the regulational mechanism of eRNA. miRNA function in osteogenesis was elucidated by miR mimic and inhibitor transfection experiments.

Results: miR-3129 was found to be located adjacent in a SE (osteoblast-specific SE_46171) specifically activated in osteoblasts by in silico analysis. A RT-quantitative PCR analysis of human bone marrow-derived MSC (hBMSC) cells showed that eRNA_2S was transcribed from the SE with the expression of miR-3129. Knockdown of eRNA_2S by locked nucleic acid as well as treatment of SE inhibitors JQ1 or THZ1 resulted in low miR-3129 levels. Overexpression of miR-3129 promoted hBMSC osteogenesis, while knockdown of miR-3129 inhibited hBMSC osteogenesis. Solute carrier family 7 member 11 (SLC7A11), encoding a bone formation suppressor, was upregulated following miR-3129-5p inhibition and identified as a target gene for miR-3129 during differentiation of hBMSCs into osteoblasts.

Conclusions: miR-3129 expression is regulated by SEs via eRNA_2S and this miRNA promotes hBMSC differentiation into osteoblasts through downregulating the target gene SLC7A11. Thus, the present study uncovers a commitment of an eRNA via a miR-3129/SLC7A11 regulatory pathway during osteogenesis of hBMSCs.

Keywords: Enhancer RNA; Mesenchymal stem cells; Osteogenesis; Super-enhancer; miR-3129.

Fig. 1

The miR-3129 gene located nearby osteoblast-specific SE domains and relevant eRNA expression. A Schematic diagram of the in silico analysis, based on the dbSUPER database. B Two osteoblast-specific SE (SE_46171 and SE_46098) domains are located near the miR-3129 gene on chromosome 2, and eight putative eRNAs may be expressed around H3K27ac. C, D Eight types of eRNA were highly expressed during the early stage of MSC differentiation into osteoblasts. The levels of eRNA expression were quantified by qPCR. The amounts of the eRNA transcripts were expressed relative to the amount of GAPDH transcript. Data are expressed as mean ± SD from three independent experiments (each n = 3 in C and D). ***p<0.001 vs. day 1 by one-way ANOVA. N. S, not significant; RQ, relative quantification

Fig. 2

miR-3129-5p expression was suppressed by SE inhibitors in a time-dependent and dose-dependent manner. A, C, E, G eRNA expression was examined at 24h after treatment of MSCs with the indicated concentrations of JQ1 (A) and THZ1 (E) and at the indicated time points after treatment of MSCs with 60 nM JQ1 (C) and 100 nM THZ1 (G). B, D, F, H Expression of miR-3129-5p and miR-23a was examined at 24h after treatment of MSCs with the indicated concentrations of JQ1 (B) and THZ1 (F) and at the indicated time points after treatment of MSCs with 60 nM JQ1 (D) and 100 nM THZ1 (H). The levels of eRNA and miRNA expression were quantified by qPCR. The amounts of eRNA and miRNA transcripts were expressed relative to the amounts of GAPDH and U6sn transcripts, respectively. Data are expressed as mean ± SD from three independent experiments (each n = 3 in A–H). ***p<0.001 vs. Veh or 0 h by one-way ANOVA. N.S, not significant; RQ, relative quantification; Veh, vehicle (DMSO)

Fig. 3

Knockdown of eRNA_2S suppressed miR-3129-5p expression in hBMSCs. A Positions of the putative eRNA_2, eRNA_4, and eRNA_7 transcripts during MSC differentiation into osteoblasts. B, D Each sense (B) and antisense (D) eRNA was examined in MSCs transfected with antisense LNA GapmeR. C, E miR-3129-5p expression was examined in MSCs transfected with sense (C) and antisense (E) LNA GapmeRs for 24h. The expression of miR-23a was examined as a control. The levels of eRNA and miRNA expression were quantified by qPCR. The amounts of eRNA and miRNA transcripts were expressed relative to the amounts of GAPDH and U6sn transcripts, respectively. Data are expressed as mean ± SD from three independent experiments (each n = 3 in B, C, D, and E). **p = 0.001, ***p < 0.001 vs. NC by Student’s t-test and one-way ANOVA. LNA, locked nucleic acid; NC, LNA GapmeR-negative control; S, sense; AS, antisense; RQ, relative quantification

Fig. 4

miR-3129 was expressed in the early stage of osteoblast differentiation and enhanced osteogenesis of hBMSCs. A Sequences and structure of human hsa-miR-3129, based on the miRBase database. B miR-3129 expression was examined at the indicated time points during osteogenesis of MSCs. The levels of miRNA expression were quantified by qPCR. The amounts of miRNA transcripts were expressed relative to the amount of U6sn transcript. Data are expressed as mean ± SD from three independent experiments. C Calcification was examined in MSCs and NHDF cells treated with Pre-miR-3129-5p and Pre-miR-NC by Alizarin Red S staining. Representative data are provided from three independent experiments (each n = 3 in B and C). *p<0.05, ***p<0.001 vs. day 1 by one-way ANOVA. N.S, not significant; RQ, relative quantification; OB, osteoblast. NHDF, normal human dermal fibroblast

Fig. 5

miR-3129-5p mimic transfection enhanced hBMSC osteoblast differentiation through downregulating SLC7A11. A Massive screening for miR-3129-5p putative target genes using three different databases. B–D Expression of miR-3129 (B), SLC7A11 (C), and osteoblast markers (D) in MSCs at 48h (B, C) and day 18 (D) after transfection with miR-3129-5p and miR-3129-3p mimics. The levels of miRNA and mRNA expression were quantified by qPCR. The amounts of miRNA and mRNA transcripts were expressed relative to the amounts of U6sn and GAPDH transcripts, respectively. E, F Alizarin Red S and alkaline phosphatase staining were performed in three independent experiments at day 21 after transfection of miR-3129 mimics (E), followed by quantification with ImageJ software (F). Data are expressed as mean ± SD from three independent experiments (each n=3 in B, C, D, E, and F). *p < 0.05, **p = 0.001, ***p < 0.001 vs. NC mimic by Student’s t-test. NC, negative control; OB, osteoblast; RQ, relative quantification

Fig. 6

Inhibition of miR-3129-5p attenuated osteogenesis of hBMSCs. A–C Expression of miR-3129 (A), SLC7A11 mRNA and protein (B), and osteoblast markers (C) in MSCs at 48h (A, B) and day 18 (C) after transfection of miR-3129-5p and miR-3129-3p inhibitors. The levels of miRNA and mRNA expression were quantified by qPCR. The amounts of miRNA and mRNA transcripts were expressed relative to the amounts of U6sn and GAPDH transcripts, respectively. B The levels of SLC7A11 protein were detected by Western blotting. β-actin was used as a loading control. The images cropped from the data (n=1) on Fig. S4 are shown on the right side of Fig. 6B (see Additional file 7). D, E Alizarin Red S and alkaline phosphatase staining were performed in three independent experiments on day 21 after transfection of miRNA inhibitors (D), followed by quantification with ImageJ software (E). Western blotting was performed one time (n = 1 in B). Data are expressed as mean ± SD from three independent experiments (each n = 3 in A–H). *p<0.05, ***p<0.001 vs. NC inhibitor by Student’s t-test. NC, negative control; RQ, relative quantification