miR‑488 negatively regulates osteogenic differentiation of bone marrow mesenchymal stem cells induced by psoralen by targeting Runx2

Abstract

It has been previously reported that psoralen, one of the active ingredients in Psoralea corylifolia, could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), suggesting its potential to treat osteoporosis. Additionally, runt‑related transcription factor 2 (Runx2) is a transcription factor that plays vital roles in BMSC osteogenic differentiation. However, whether and how microRNAs (miRNAs/miRs) modulate osteogenic differentiation induced by psoralen have not yet been examined, to the best of the authors' knowledge. The present study aimed to identify the miRNA target genes that regulate osteogenic differentiation of BMSCs induced by psoralen. A Cell Counting Kit‑8 assay and alizarin red staining were used to detect the viability and osteogenic differentiation of BMSCs, respectively, under treatment with psoralen. miRNA microarray analysis was performed to identify the differentially expressed miRNAs under treatment with psoralen. A bioinformatics analysis and a luciferase reporter assay were conducted to identify the targets of miR‑488. Finally, the mechanisms of miR‑488 in psoralen‑induced BMSC osteogenic differentiation were investigated using overexpression or inhibition methods in vitro. Cell viability was elevated and osteogenic differentiation of BMSCs was improved under treatment with psoralen. miRNA microarray analysis and further validation by reverse transcription‑quantitative PCR revealed that miR‑488 was downregulated during psoralen‑induced BMSC osteogenic differentiation. Bioinformatics analysis and experimental validation by a luciferase reporter assay identified Runx2 as a potential target of miR‑488. Overexpression of miR‑488 by transfection with miR‑488 mimics markedly inhibited the expression of Runx2, Osterix and alkaline phosphatase, whereas, the inhibition of miR‑488 expression by the miR‑488 inhibitor promoted their expression compared with the control. Rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR‑488 on BMSC osteogenic differentiation. The present results suggested that miR‑488 is a negative regulator of psoralen‑induced BMSC osteogenic differentiation by targeting Runx2, providing a possible therapeutic target for osteoporosis.

Figure 1.

Psoralen promotes the viability and osteogenic differentiation of BMSCs. (A) Cell Counting Kit-8 assay showed that significantly elevated cell viability was found in BMSCs treated with psoralen at all time points except at 12 h. (B) Alizarin red staining (magnification, ×200) showed negligible calcium mineral deposition in the blank control group, while similar calcium mineral deposition was observed in the psoralen and positive control groups. *P<0.05 vs. control. BMSCs, bone marrow mesenchymal stem cells; OD, optical density.

Figure 2.

Heat map and bar graph of differentially expressed miRs during the psoralen-induced osteogenic differentiation of bone marrow mesenchymal stem cells. (A) Compared with the control group, 91 miRs were differentially expressed, among which miR-488 was significantly downregulated. Red indicates upregulation and green indicates downregulation. (B) Reverse transcription-quantitative PCR showed that miR-122, miR-154 and miR-488 were downregulated, while miR-205 was upregulated, compared with the control group. *P<0.05 vs. the respective control group. miR, microRNA.

Figure 3.

Runx2 is a potential target of miR-488. (A) Construction profile of the pLUC-Runx2 vector is presented in the diagram, which contained the miR-488 target sites in Runx2 3′UTR-wt, red sequence presented the mutation region. Reverse transcription-quantitative PCR showed that (B) miR-488 mimics and (C) miR-488 inhibitor were successfully transfected into cells. (D) Reverse transcription-quantitative PCR showed cotransfection of miR-488 mimics and miR-488 inhibitor. Reverse transcription-quantitative PCR showed that (E) pcDNA 3.1-Runx2 was successfully transfected into cells. *P<0.05. (F) Relative luciferase activity demonstrated that Runx2 is a potential target of miR-488. *P<0.05 vs. the miR control group; ^#P<0.05 vs. the miR-488 mimics group. Runx2, runt-related transcription factor 2; miR, microRNA; 3′UTR, 3′-untranslated region; wt, wild-type; mut, mutant; ns, not significant.

Figure 4.

miR-488 negatively regulates the osteogenic differentiation of bone marrow mesenchymal stem cells. (A) mRNA expression levels of Runx2, Osterix and ALP were decreased in the miR-488 mimics group and were increased in the miR-488 inhibitor group compared with the control group. Protein expression levels of Runx2, Osterix and ALP were decreased in the miR-488 mimics group and were increased in the miR-488 inhibitor group compared with the control group by (B) western blot analysis and (C) subsequent densitometry. (D) Alizarin red staining (magnification, ×100) showed that more calcium mineral deposition was found in the miR-488 inhibitor group than in the control and miR-488 mimics groups. White arrows indicate calcium mineral deposition. (E) Immunocytochemistry (magnification, ×200) demonstrated that the expression levels of Runx2, Osterix and ALP in the miR-488 mimics group were notably lower than in the miR-488 inhibitor group. *P<0.05 vs. the respective miR control group. miR, microRNA; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase.

Figure 5.

Runx2 overexpression partially rescues the inhibitory effect of miR-488 on the osteogenic differentiation of BMSCs. (A) AR-S indicated that more calcium mineral deposition was found in the psoralen+pcDNA 3.1-Runx2+miR-488 mimics group than in the psoralen+miR-488 mimics group, while the calcium mineral deposition in the psoralen+pcDNA 3.1-Runx2+miR-488 mimics group was less than that in the psoralen+pcDNA 3.1-Runx2 group. White arrows indicate calcium mineral deposition. (B) Western blotting and (C) subsequent densitometry, and (D) immunocytochemistry detected the protein expression of osteogenic-specific factors in BMSCs transfected with miR-488 mimics and pcDNA 3.1-Runx2, which was similar to the AR-S results. *P<0.05 vs. the psoralen+miR-488 mimics group. miR, microRNA; BMSCs, bone marrow mesenchymal stem cells; Runx2, runt-related transcription factor 2; AR-S, alizarin red staining.