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miR-1827 inhibits osteogenic differentiation by targeting IGF1 in MSMSCs

View Article: PubMed Central - PubMed

ABSTRACT

We recently reported that maxillary sinus membrane stem cells (MSMSCs) have osteogenic potential. However, the biological mechanisms of bone formation remain unclear. In this study, we investigated the role and mechanisms of microRNAs (miRNAs) in the osteogenic differentiation of MSMSCs. The expression of miRNAs was determined in differentiated MSMSCs by comprehensive miRNA microarray analysis and quantitative RT-PCR (qRT-PCR). We selected miR-1827 for functional follow-up studies to explore its significance in MSMSCs. Here, miR-1827 was found to be up-regulated during osteogenic differentiation of MSMSCs. Over expression of miR-1827 inhibited osteogenic differentiation of MSMSCs in vitro, whereas the repression of miR-1827 greatly promoted cell differentiation. Further experiments confirmed that insulin-like growth factor 1 (IGF1) is a direct target of miR-1827. miR-1827 inhibited osteogenic differentiation partially via IGF1, which in turn is a positive regulator of osteogenic differentiation. Moreover, miR-1827 suppressed ectopic bone formation and silencing of miR-1827 led to increased bone formation in vivo. In summary, this study is the first to demonstrate that miR-1827 can regulate osteogenic differentiation. The increase in miR-1827 expression observed during osteogenesis is likely a negative feedback mechanism, thus offering a potential therapeutic target to address inadequate bone volume for dental implantation through inhibiting miR-1827.

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Related in: MedlinePlus

miR-1827 inhibits osteogenic differentiation in vitro.To evaluate the effects of miR-1827 on osteogenic differentiation, MSMSCs and BMSSCs were transfected with a miR-1827 mimic (mimic-1827), miR-1827 inhibitor (inhibitor-1827) or their respective negative controls (mimic-NC, inhibitor-NC). (a) Representative fluorescent images of MSMSCs transfected with a miRNA nucleoside analogue for 24 h. Fluorescence indicated transfected cells. (b,c) qRT-PCR analysis of osteoblastic marker (Runx2 and OPN) mRNA expression after 48 h of osteogenic induction. The gene expression levels in MSMSCs transfected with the respective miRNA negative controls were set as the control (normalized to GAPDH). (d) ALP activity in MSMSCs at 48 h. (e,f) Western blot analysis of Runx2 and OPN protein expression in MSMSCs after 48 h of osteogenic induction. GAPDH was used to assess the amount of protein loaded per sample. (g) The formation of mineralized nodules in MSMSCs was observed by Alizarin Red staining. Cells were cultured in osteogenic induction medium for 4 weeks. (h) The mineralized nodules in different groups were quantified using cetylpyridinium chloride. OD, optical density. (i,j,k) Alterations in osteoblastic marker (Runx2 and OPN) mRNA expression and ALP activity were observed in BMSSCs by qRT-PCR and an ALP activity assay. For each group, values are the mean ± SD; n = 3, *P < 0.05, **P < 0.01. NS, not significant. Scar bars represent 100 μm.
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f2: miR-1827 inhibits osteogenic differentiation in vitro.To evaluate the effects of miR-1827 on osteogenic differentiation, MSMSCs and BMSSCs were transfected with a miR-1827 mimic (mimic-1827), miR-1827 inhibitor (inhibitor-1827) or their respective negative controls (mimic-NC, inhibitor-NC). (a) Representative fluorescent images of MSMSCs transfected with a miRNA nucleoside analogue for 24 h. Fluorescence indicated transfected cells. (b,c) qRT-PCR analysis of osteoblastic marker (Runx2 and OPN) mRNA expression after 48 h of osteogenic induction. The gene expression levels in MSMSCs transfected with the respective miRNA negative controls were set as the control (normalized to GAPDH). (d) ALP activity in MSMSCs at 48 h. (e,f) Western blot analysis of Runx2 and OPN protein expression in MSMSCs after 48 h of osteogenic induction. GAPDH was used to assess the amount of protein loaded per sample. (g) The formation of mineralized nodules in MSMSCs was observed by Alizarin Red staining. Cells were cultured in osteogenic induction medium for 4 weeks. (h) The mineralized nodules in different groups were quantified using cetylpyridinium chloride. OD, optical density. (i,j,k) Alterations in osteoblastic marker (Runx2 and OPN) mRNA expression and ALP activity were observed in BMSSCs by qRT-PCR and an ALP activity assay. For each group, values are the mean ± SD; n = 3, *P < 0.05, **P < 0.01. NS, not significant. Scar bars represent 100 μm.

Mentions: To study the biological role of miR-1827 in osteogenic differentiation, MSMSCs were transfected with a miR-1827 inhibitor or miR-1827 mimic to alter the expression levels of miR-1827 in vitro (Fig. 2a). The effects of miR-1827 inhibitor or mimic on osteogenic differentiation were evaluated by observing mineralized nodule formation, alkaline phosphatase (ALP) activity and the expression levels of Runx2 and osteopontin (OPN). After transfection with miR-1827 mimic, the mRNA expression levels of osteogenic-specific markers (Runx2 and OPN) decreased compared with the control group as indicated by qRT-PCR (Fig. 2b,c). In addition, ALP activity and the protein expression levels of Runx2 and OPN decreased (Fig. 2d,e,f). Furthermore, the formation of mineralized nodules was also reduced (Fig. 2g,h), implying that over expression of miR-1827 inhibited osteogenic differentiation. On the contrary, Runx2 and OPN mRNA expression (Fig. 2b,c), ALP activity (Fig. 2d), and protein levels of Runx2 and OPN (Fig. 2e,f) were all increased following transfection with the miR-1827 inhibitor. Furthermore, the formation of mineralized nodules was also increased (Fig. 2g,h), implying that the repression of miR-1827 promoted osteogenic differentiation. To further validate the role of miR-1827 in osteogenic differentiation, the miR-1827 mimic and inhibitor were transfected into BMSSCs. After 48 h of osteogenic induction, alterations in Runx2 and OPN mRNA expression and ALP activity were observed to be similar to those detected in MSMSCs, as indicated by qRT-PCR and an ALP activity assay (Fig. 2i,j,k). These results indicate that miR-1827 inhibits osteogenic differentiation.


miR-1827 inhibits osteogenic differentiation by targeting IGF1 in MSMSCs
miR-1827 inhibits osteogenic differentiation in vitro.To evaluate the effects of miR-1827 on osteogenic differentiation, MSMSCs and BMSSCs were transfected with a miR-1827 mimic (mimic-1827), miR-1827 inhibitor (inhibitor-1827) or their respective negative controls (mimic-NC, inhibitor-NC). (a) Representative fluorescent images of MSMSCs transfected with a miRNA nucleoside analogue for 24 h. Fluorescence indicated transfected cells. (b,c) qRT-PCR analysis of osteoblastic marker (Runx2 and OPN) mRNA expression after 48 h of osteogenic induction. The gene expression levels in MSMSCs transfected with the respective miRNA negative controls were set as the control (normalized to GAPDH). (d) ALP activity in MSMSCs at 48 h. (e,f) Western blot analysis of Runx2 and OPN protein expression in MSMSCs after 48 h of osteogenic induction. GAPDH was used to assess the amount of protein loaded per sample. (g) The formation of mineralized nodules in MSMSCs was observed by Alizarin Red staining. Cells were cultured in osteogenic induction medium for 4 weeks. (h) The mineralized nodules in different groups were quantified using cetylpyridinium chloride. OD, optical density. (i,j,k) Alterations in osteoblastic marker (Runx2 and OPN) mRNA expression and ALP activity were observed in BMSSCs by qRT-PCR and an ALP activity assay. For each group, values are the mean ± SD; n = 3, *P < 0.05, **P < 0.01. NS, not significant. Scar bars represent 100 μm.
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f2: miR-1827 inhibits osteogenic differentiation in vitro.To evaluate the effects of miR-1827 on osteogenic differentiation, MSMSCs and BMSSCs were transfected with a miR-1827 mimic (mimic-1827), miR-1827 inhibitor (inhibitor-1827) or their respective negative controls (mimic-NC, inhibitor-NC). (a) Representative fluorescent images of MSMSCs transfected with a miRNA nucleoside analogue for 24 h. Fluorescence indicated transfected cells. (b,c) qRT-PCR analysis of osteoblastic marker (Runx2 and OPN) mRNA expression after 48 h of osteogenic induction. The gene expression levels in MSMSCs transfected with the respective miRNA negative controls were set as the control (normalized to GAPDH). (d) ALP activity in MSMSCs at 48 h. (e,f) Western blot analysis of Runx2 and OPN protein expression in MSMSCs after 48 h of osteogenic induction. GAPDH was used to assess the amount of protein loaded per sample. (g) The formation of mineralized nodules in MSMSCs was observed by Alizarin Red staining. Cells were cultured in osteogenic induction medium for 4 weeks. (h) The mineralized nodules in different groups were quantified using cetylpyridinium chloride. OD, optical density. (i,j,k) Alterations in osteoblastic marker (Runx2 and OPN) mRNA expression and ALP activity were observed in BMSSCs by qRT-PCR and an ALP activity assay. For each group, values are the mean ± SD; n = 3, *P < 0.05, **P < 0.01. NS, not significant. Scar bars represent 100 μm.
Mentions: To study the biological role of miR-1827 in osteogenic differentiation, MSMSCs were transfected with a miR-1827 inhibitor or miR-1827 mimic to alter the expression levels of miR-1827 in vitro (Fig. 2a). The effects of miR-1827 inhibitor or mimic on osteogenic differentiation were evaluated by observing mineralized nodule formation, alkaline phosphatase (ALP) activity and the expression levels of Runx2 and osteopontin (OPN). After transfection with miR-1827 mimic, the mRNA expression levels of osteogenic-specific markers (Runx2 and OPN) decreased compared with the control group as indicated by qRT-PCR (Fig. 2b,c). In addition, ALP activity and the protein expression levels of Runx2 and OPN decreased (Fig. 2d,e,f). Furthermore, the formation of mineralized nodules was also reduced (Fig. 2g,h), implying that over expression of miR-1827 inhibited osteogenic differentiation. On the contrary, Runx2 and OPN mRNA expression (Fig. 2b,c), ALP activity (Fig. 2d), and protein levels of Runx2 and OPN (Fig. 2e,f) were all increased following transfection with the miR-1827 inhibitor. Furthermore, the formation of mineralized nodules was also increased (Fig. 2g,h), implying that the repression of miR-1827 promoted osteogenic differentiation. To further validate the role of miR-1827 in osteogenic differentiation, the miR-1827 mimic and inhibitor were transfected into BMSSCs. After 48 h of osteogenic induction, alterations in Runx2 and OPN mRNA expression and ALP activity were observed to be similar to those detected in MSMSCs, as indicated by qRT-PCR and an ALP activity assay (Fig. 2i,j,k). These results indicate that miR-1827 inhibits osteogenic differentiation.

View Article: PubMed Central - PubMed

ABSTRACT

We recently reported that maxillary sinus membrane stem cells (MSMSCs) have osteogenic potential. However, the biological mechanisms of bone formation remain unclear. In this study, we investigated the role and mechanisms of microRNAs (miRNAs) in the osteogenic differentiation of MSMSCs. The expression of miRNAs was determined in differentiated MSMSCs by comprehensive miRNA microarray analysis and quantitative RT-PCR (qRT-PCR). We selected miR-1827 for functional follow-up studies to explore its significance in MSMSCs. Here, miR-1827 was found to be up-regulated during osteogenic differentiation of MSMSCs. Over expression of miR-1827 inhibited osteogenic differentiation of MSMSCs in vitro, whereas the repression of miR-1827 greatly promoted cell differentiation. Further experiments confirmed that insulin-like growth factor 1 (IGF1) is a direct target of miR-1827. miR-1827 inhibited osteogenic differentiation partially via IGF1, which in turn is a positive regulator of osteogenic differentiation. Moreover, miR-1827 suppressed ectopic bone formation and silencing of miR-1827 led to increased bone formation in vivo. In summary, this study is the first to demonstrate that miR-1827 can regulate osteogenic differentiation. The increase in miR-1827 expression observed during osteogenesis is likely a negative feedback mechanism, thus offering a potential therapeutic target to address inadequate bone volume for dental implantation through inhibiting miR-1827.

No MeSH data available.


Related in: MedlinePlus