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miR-23a impairs bone differentiation in osteosarcoma via down-regulation of GJA1.

Gindin Y, Jiang Y, Francis P, Walker RL, Abaan OD, Zhu YJ, Meltzer PS - Front Genet (2015)

Bottom Line: Experimental results demonstrate that over-expression of miR-23a delays differentiation in this system.Analysis of gene expression data, housed at Gene Expression Omnibus, reveals that Cx43 is consistently up-regulated during osteoblast differentiation.Suppression of Cx43 mRNA by miR-23a was confirmed in vitro using a luciferase reporter assay.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Center for Cancer Research, National Institutes of Health Bethesda, MD, USA ; Graduate Program in Bioinformatics, Boston University Boston, MA, USA.

ABSTRACT
Osteosarcoma is the most common type of bone cancer in children and adolescents. Impaired differentiation of osteoblast cells is a distinguishing feature of this aggressive disease. As improvements in survival outcomes have largely plateaued, better understanding of the bone differentiation program may provide new treatment approaches. The miRNA cluster miR-23a~27a~24-2, particularly miR-23a, has been shown to interact with genes important for bone development. However, global changes in gene expression associated with functional gain of this cluster have not been fully explored. To better understand the relationship between miR-23a expression and bone cell differentiation, we carried out a large-scale gene expression analysis in HOS cells. Experimental results demonstrate that over-expression of miR-23a delays differentiation in this system. Downstream bioinformatic analysis identified miR-23a target gene connexin-43 (Cx43/GJA1), a mediator of intercellular signaling critical to osteoblast development, as acutely affected by miR-23a levels. Connexin-43 is up-regulated in the course of HOS cell differentiation and is down-regulated in cells transfected with miR-23a. Analysis of gene expression data, housed at Gene Expression Omnibus, reveals that Cx43 is consistently up-regulated during osteoblast differentiation. Suppression of Cx43 mRNA by miR-23a was confirmed in vitro using a luciferase reporter assay. This work demonstrates novel interactions between microRNA expression, intercellular signaling and bone differentiation in osteosarcoma.

No MeSH data available.


Related in: MedlinePlus

Alizarin red staining of HOS cells. (A) Alizarin red staining of HOS cells during the differentiation time course. Red staining is indicative of calcium deposits. (B) Untreated cells.
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Figure 1: Alizarin red staining of HOS cells. (A) Alizarin red staining of HOS cells during the differentiation time course. Red staining is indicative of calcium deposits. (B) Untreated cells.

Mentions: To confirm that HOS cells are amendable to bone differentiation induction (Siggelkow et al., 1998), we treated these cells with L-ascorbic acid, which induces the formation of collagenous extracellular matrix and brings an osteoblast-specific gene expression program in osteogenic lineage cells (Franceschi et al., 1994). We then monitored HOS cell culture for the presence of calcium deposits, which serve as a marker of bone mineralization, via Alizarin Red staining. Our results indicate that HOS cells undergo osteoblast-like differentiation upon stimulation with L-ascorbic acid. HOS cells exhibit intense Alizarin Red staining on day 7 post differentiation induction (Figure 1). We confirmed this result by monitoring the expression of collagen Ia1 (COL1A1)—a gene marker of bone differentiation (Figure 2). We have observed a two-fold increase in COL1A1 mRNA levels between the initial and terminal differentiation time-points.


miR-23a impairs bone differentiation in osteosarcoma via down-regulation of GJA1.

Gindin Y, Jiang Y, Francis P, Walker RL, Abaan OD, Zhu YJ, Meltzer PS - Front Genet (2015)

Alizarin red staining of HOS cells. (A) Alizarin red staining of HOS cells during the differentiation time course. Red staining is indicative of calcium deposits. (B) Untreated cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488756&req=5

Figure 1: Alizarin red staining of HOS cells. (A) Alizarin red staining of HOS cells during the differentiation time course. Red staining is indicative of calcium deposits. (B) Untreated cells.
Mentions: To confirm that HOS cells are amendable to bone differentiation induction (Siggelkow et al., 1998), we treated these cells with L-ascorbic acid, which induces the formation of collagenous extracellular matrix and brings an osteoblast-specific gene expression program in osteogenic lineage cells (Franceschi et al., 1994). We then monitored HOS cell culture for the presence of calcium deposits, which serve as a marker of bone mineralization, via Alizarin Red staining. Our results indicate that HOS cells undergo osteoblast-like differentiation upon stimulation with L-ascorbic acid. HOS cells exhibit intense Alizarin Red staining on day 7 post differentiation induction (Figure 1). We confirmed this result by monitoring the expression of collagen Ia1 (COL1A1)—a gene marker of bone differentiation (Figure 2). We have observed a two-fold increase in COL1A1 mRNA levels between the initial and terminal differentiation time-points.

Bottom Line: Experimental results demonstrate that over-expression of miR-23a delays differentiation in this system.Analysis of gene expression data, housed at Gene Expression Omnibus, reveals that Cx43 is consistently up-regulated during osteoblast differentiation.Suppression of Cx43 mRNA by miR-23a was confirmed in vitro using a luciferase reporter assay.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Center for Cancer Research, National Institutes of Health Bethesda, MD, USA ; Graduate Program in Bioinformatics, Boston University Boston, MA, USA.

ABSTRACT
Osteosarcoma is the most common type of bone cancer in children and adolescents. Impaired differentiation of osteoblast cells is a distinguishing feature of this aggressive disease. As improvements in survival outcomes have largely plateaued, better understanding of the bone differentiation program may provide new treatment approaches. The miRNA cluster miR-23a~27a~24-2, particularly miR-23a, has been shown to interact with genes important for bone development. However, global changes in gene expression associated with functional gain of this cluster have not been fully explored. To better understand the relationship between miR-23a expression and bone cell differentiation, we carried out a large-scale gene expression analysis in HOS cells. Experimental results demonstrate that over-expression of miR-23a delays differentiation in this system. Downstream bioinformatic analysis identified miR-23a target gene connexin-43 (Cx43/GJA1), a mediator of intercellular signaling critical to osteoblast development, as acutely affected by miR-23a levels. Connexin-43 is up-regulated in the course of HOS cell differentiation and is down-regulated in cells transfected with miR-23a. Analysis of gene expression data, housed at Gene Expression Omnibus, reveals that Cx43 is consistently up-regulated during osteoblast differentiation. Suppression of Cx43 mRNA by miR-23a was confirmed in vitro using a luciferase reporter assay. This work demonstrates novel interactions between microRNA expression, intercellular signaling and bone differentiation in osteosarcoma.

No MeSH data available.


Related in: MedlinePlus