eRNAs from super-enhancers located near the miR-3129 gene region are highly expressed in the early stage of hBMSC osteogenesis
SEs have been reported to determine cell identity by inducing cell differentiation [8]. To identify novel SE-associated genes that can induce osteoblast differentiation, we performed in silico mining to search for potential osteogenic genes regulated by SEs that are specifically activated in osteoblasts (Fig. 1A). Firstly, we focused on 1089 genes regulated by SE activated in osteoblasts through dbSuper. Next, we extracted many genes activated in 101 human tissues/cell types except osteoblasts, screened 1044 genes overlapped with osteoblast, and excluded 1044 common genes of 1089 genes activated in osteoblast. As a result, we found 45 genes (including 7 non-coding RNAs) specifically regulated by SE in osteoblasts (Supplementary Table S3 (see Additional file 3)). Our interest was to study the regulatory mechanism and role of a non-coding RNA which remains to be defined. Therefore, we attempted to find a candidate non-coding gene which had proximal and extragenic (gene-free) SEs. Finally, miR-3129 was extracted as a candidate gene. The miR-3129 locus has a SE structure (SE_45650 registered in dbSUPER) located in an intragenic region and two SEs (SE_46171 and SE_46098) located in the proximal extragenic region (Fig. 1B). Within these regions, there are several peaks for histone H3 containing acetylated lysine 27 (H3K27ac), a SE marker, in human skeletal muscle myoblasts. We picked up 8 regions of the registered high H3K27ac peaks on the database and assumed these regions transcribe eRNAs in these 2 SE domains, given that eRNAs are defined as non-coding RNAs transcribed from SE domains encompassing layer(s). Therefore, we performed RT-qPCR and then found at least 8 types of eRNAs in 8 different regions within SE_46171 and SE_46098 in the hBMSCs cultured with a growth medium and an osteoblast induction medium (Fig. 1C, D). The hBMSCs that have just adhered to the plate might be unstable in gene expression profiles, presumably showing higher expression in the case of eRNAs. These eRNAs were most highly expressed in the SE domains during the early stage of osteoblast differentiation, and their expression gradually decreased as differentiation progressed (Fig. 1D).
eRNAs and miR-3129 expression were suppressed by SE inhibitor treatment in a time-dependent and dose-dependent manner
In order to determine the involvement of eRNAs in miR-3129 expression, we attempted to suppress eRNA level by SE inhibitors and then the effect of eRNA on miR-3129 expression. The transcriptional co-activators bromodomain-containing protein 4 (BRD4) and cyclin-dependent kinase 7 (CDK7), which are responsible for SE formation, are abundantly localized in SEs which express eRNAs. Therefore, we focused on JQ1 and THZ1 as SE inhibitors, which specifically inhibit BRD4 and CDK7 activity, respectively, and then examined the effect of SE inhibitors on miR-3129 levels as well as eRNA levels. At first, hBMSCs were stimulated with various concentrations of JQ1 (60 nM and 250 nM) for 24h. The eRNAs and miR-3129 expression were then determined by RT-qPCR. SEs are generally cell-type-specific and can function as cis-regulatory elements for nearby genes. Therefore, miR-23a, a miRNA highly expressed in the early stage of hBMSC osteogenesis [26], was used as a control miRNA for miR-3129 in these experiments. As a result, the expression of each eRNA was significantly suppressed in a dose-dependent manner (Fig. 2A). The expression of miR-3129, but not miR-23a, was also suppressed (left side and right side of Fig. 2B). Next, hBMSCs were stimulated with JQ1 at a concentration of 60 nM for specified time points (0h, 6h, 12h, and 24h). The expression of each eRNA (Fig. 2C) as well as miR-3129, but not miR-23a (left side and right side of Fig. 2D), was significantly suppressed in a time-dependent manner (Fig. 2C, D). Similarly, hBMSCs were stimulated with various concentrations of THZ1 (50 nM and 100 nM) for 24h, and the expression of each eRNA (Fig. 2E) as well as miR-3129, but not miR-23a (left side and right side of Fig. 2F), was significantly suppressed in a dose-dependent manner. Furthermore, when hBMSCs were stimulated with THZ1 at a concentration of 100 nM for specified time points (0h, 6h, 12h, and 24h), the expression of each eRNA (Fig. 2G) as well as miR-3129, but not miR-23a (left side and right side of Fig. 2H), was suppressed in a time-dependent manner. These results indicated that miR-3129 expression was suppressed by SE inhibitor treatment in a time-dependent and dose-dependent manner.
Knockdown of eRNA_2S resulted in suppressing miR-3129 expression
In general, eRNAs play roles in the induction of their target gene expression. Therefore, we examined the expression of miR-3129 after eRNA knockdown. We randomly chose eRNA 2, 4, and 7 as the candidate eRNAs to investigate the roles of individual eRNAs including sense (S) and antisense (AS) eRNAs (Fig. 3A). Locked nucleic acid (LNA) sequences designed to target each eRNA and a LNA-negative control (LNA-NC) sequence were transfected into hBMSCs (Supplementary Table S2 (see Additional file 2)). The knockdown efficiency of various sense eRNAs and miR-3129 expression were examined by RT-qPCR (Fig. 3B). The expression of miR-3129 was only significantly decreased in MSCs after knockdown of eRNA_2S (transcribed from SE_46171) (Fig. 3C). The knockdown efficiency of various antisense eRNAs was also elucidated (Fig. 3D). However, the expression of miR-3129 was not altered after knockdown of any antisense eRNAs (Fig. 3E). Collectively, only knockdown of eRNA_2S showed an effective impact on miR-3129 expression.
miR-3129 expression was downregulated during osteoblast differentiation and pre-miR-3129 precursor transfection enhanced hBMSC osteogenesis
The secondary structure of the human hsa-miR-3129 precursor from miRBase (a miRNA database [27]) is shown in Fig. 4A. The expression pattern of miR-3129 during hBMSC differentiation into osteoblasts was examined by RT-qPCR. hBMSCs expressed both the 5p and 3p strands of miR-3129 regardless of the cell proliferation time (day 1, 7, and 18), and the expression levels remained unchanged (Fig. 4B). When hBMSCs were cultured in an osteoblast induction medium (OIM), the 5p and 3p strands both showed the highest expression during the early stage (day 1), and their expression gradually decreased as osteogenesis progressed (day 7 and 18). As controls for OIM, hBMSCs were cultured in adipocyte and chondrocyte induction media. The results show that expression levels of the 5p and 3p strands remained unaltered during the respective differentiation processes (Supplementary Fig. S1 (see Additional file 4)), indicating the specific role of miR-3129 in osteogenesis.
We transfected hBMSCs with a Pre-miRNA-3129 precursor and a Pre-miRNA-negative control precursor (Pre-miR-NC) to further clarify the miR-3129 function in the osteoblast differentiation process. In OIM culture, the cells showed positive Alizarin Red S (ARS) staining on day 21 (Fig. 4C). Normal human dermal fibroblast (NHDF) cells, as a control cell line, were also transfected with these precursors. Interestingly, hBMSCs transfected with the Pre-miR-3129 precursor showed stronger positivity on day 21 compared with those transfected with the Pre-miR-NC. NHDF cells were negative for ARS staining regardless of the culture medium and regardless of which miRNA precursor was transfected.
miR-3129 overexpression promoted osteoblast differentiation of hBMSCs through downregulating SLC7A11
Using three public databases (DIANA Tools microT-CDS, miRDB, and Target Scan v7.2), we extracted three groups of genes predicted to be the target of this miRNA (Fig. 5A). As a result of in silico analyses, 158 overlapping genes were found. We further divided them into 10 genes that promote osteogenesis and 10 genes that suppress osteoblast differentiation. Our previous results showed that miR-3129 has the potency to enhance osteoblast differentiation (Fig. 4C); therefore, finding a target for this miRNA will be focused on 10 genes that are suppressors of osteogenesis. hBMSCs were transfected with the miR-NC mimic, miR-3129-5p mimic, and miR-3129-3p mimic, and high levels of the 5p and 3p strands were detected, respectively, after 48h (Fig. 5B). Compared with transfection of the miR-NC mimic, transfection of the 5p mimic only decreased the expression of solute carrier family 7 member 11 (SLC7A11) among the 10 osteogenic suppressor genes (Fig. 5A), while transfection of the 3p mimic did not affect the expression of any candidate genes in mRNA levels (Fig. 5C and Supplementary Fig. S2A (see Additional file 5)). The expression of osteoblast transcription factor and marker genes such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase gene (ALPL), bone gamma-carboxyglutamate protein (BGLAP), and secrected phosphoprotein 1 (SPP1) were significantly increased by the transfection of the 5p mimic, but not the miR-NC mimic and 3p mimic (Fig. 5D). ARS and alkaline phosphatase (ALP) staining were performed to evaluate the phenotype after osteogenesis (Fig. 5E). Transfection of the miR-NC mimic and 3p mimic resulted in similar levels of positive staining for ARS and ALP, whereas transfection of the 5p mimic significantly increased both staining levels (Fig. 5F).
Knockdown of miR-3129 resulted in suppressed osteoblast differentiation of hBMSCs
Next, we evaluated the effect of knocking down the 5p and 3p strands of miR-3129 on hBMSC osteoblast differentiation with specific inhibitors. At 48h after transfection of hBMSCs with the miR-NC, miR-5p, and miR-3p inhibitors, we found that the expression of the 5p and 3p strands was suppressed (Fig. 6A). Compared with hBMSCs treated with the miR-NC inhibitor, those treated with the 5p inhibitor displayed an upregulated expression of only SLC7A11 mRNA and protein, but not mRNAs of other osteogenic suppressor genes (Fig. 5A, Fig. 6B, and Supplementary Fig. S2B (see Additional file 5)). In contrast, those treated with the 3p inhibitor showed no such upregulation in SLC7A11 mRNA and protein level (Fig. 6B) as well as mRNAs of other osteogenic suppressor genes (Fig. 5A and Supplementary Fig. S2B (see Additional file 5)). Meanwhile, there were no differences in the expression levels of a series of osteoblast transcription factor and marker genes between the miR-NC inhibitor and 3p inhibitor, while the 5p inhibitor significantly suppressed the expression of these genes (Fig. 6C). Staining for ARS as well as ALP was performed to evaluate the phenotype after osteogenesis (Fig. 6D). Treatment with the miR-NC and 3p inhibitors resulted in similar levels of positive staining for ARS and ALP, while treatment with the 5p strand inhibitor significantly suppressed both staining levels (Fig. 6E).
SLC7A11 expression enhanced after eRNA_2S knockdown in hBMSCs
In our previous results, SLC7A11 was identified as the miR-3129 target gene in hBMSCs (Figs. 5 and 6). We further examined the involvement of eRNAs in the regulation of SLC7A11 expression and found that only knockdown of eRNA_2S significantly increased the SLC7A11 level (Supplementary Fig. S3 (see Additional file 6)). This result was consistent with our previous findings and further determined the regulatory role of eRNA_2S in miR-3129 expression in hBMSCs.