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Busulfan inhibits growth of human osteosarcoma through miR-200 family microRNAs in vitro and in vivo.

Mei Q, Li F, Quan H, Liu Y, Xu H - Cancer Sci. (2014)

Bottom Line: Busulfan has been widely used to treat CML.Moreover, a series of loss-of-function and gain-of-function experiments further indicated that busulfan may have its anti-osteosarcoma effect by upregulating the microRNA-200 (miR-200) family which subsequently downregulated its target genes ZEB1 and ZEB2.Taken together, our data suggest that busulfan may have an anti-osteosarcoma effect through downregulating ZEB1 and ZEB2 through activating the miR-200 family, highlighting a possibility of using busulfan as a novel therapy for osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: 169th Hospital, School of Medicine, Hunan Normal University, Changsha, China.

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Downregulating the microRNA-200 (miR-200) family or upregulating ZEB1 and ZEB2 reversed the anti-osteosarcoma effect of busulfan. (a) U2OS cells were transfected with antisense oligonucleotides (ASO) of miR-200a, miR-200b, miR-200b, miR-141, and miR-429 to downregulate the expression of miRNAs. The percentage of viable cells, apoptosis, β-gal-positive cells and 2'-7'-Dichlorodihydrofluorescein diacetate (DCF-DA) levels were assayed after 24 h. All data were normalized to negative control (NC). (b) MG-63 cells were transfected with pcDNA3.1-ZEB1 or pcDNA3.1-ZEB2, two overexpressing plasmids. The percentage of viable cells, apoptosis, β-gal-positive cells and DCF-DA levels were assayed after 24 h. (c) Downregulation of all five members in the miR-200 family by ASO resulted in upregulation of ZEB1 and ZEB2. *P < 0.05.
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fig03: Downregulating the microRNA-200 (miR-200) family or upregulating ZEB1 and ZEB2 reversed the anti-osteosarcoma effect of busulfan. (a) U2OS cells were transfected with antisense oligonucleotides (ASO) of miR-200a, miR-200b, miR-200b, miR-141, and miR-429 to downregulate the expression of miRNAs. The percentage of viable cells, apoptosis, β-gal-positive cells and 2'-7'-Dichlorodihydrofluorescein diacetate (DCF-DA) levels were assayed after 24 h. All data were normalized to negative control (NC). (b) MG-63 cells were transfected with pcDNA3.1-ZEB1 or pcDNA3.1-ZEB2, two overexpressing plasmids. The percentage of viable cells, apoptosis, β-gal-positive cells and DCF-DA levels were assayed after 24 h. (c) Downregulation of all five members in the miR-200 family by ASO resulted in upregulation of ZEB1 and ZEB2. *P < 0.05.

Mentions: To define whether busulfan produced its anti-osteosarcoma effect through downregulation of the miRNA-200 family and by modulation of expression of its downstream targets ZEB1 and ZEB2, we inhibited the expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, or overexpressed ZEB1 and ZEB1, by miRNA antisense oligonucleotide transfection or plasmid transfection (p-ZEB1 or p-ZEB2), respectively. We found that downregulation of all five members in the miR-200 family reversed the effect induced by busulfan in terms of cell viability, proliferation, level of ROS apoptosis, and cell senescence (Fig. 3a). Similarly, we found that upregulation of ZEB1 or ZEB2 expression also reversed the anti-osteosarcoma effect of busulfan (Fig. 3b), without affecting the expression levels of the miR-200 family (data not shown). Of note, downregulation of all five members in the miR-200 family resulted in upregulation of ZEB1 and ZEB2 (Fig. 3c). Taken together, these loss-of-function experiments thus indicate that busulfan may have its anti-osteosarcoma effect by upregulating ZEB1 and ZEB2 through activating the miRNA-200 family.


Busulfan inhibits growth of human osteosarcoma through miR-200 family microRNAs in vitro and in vivo.

Mei Q, Li F, Quan H, Liu Y, Xu H - Cancer Sci. (2014)

Downregulating the microRNA-200 (miR-200) family or upregulating ZEB1 and ZEB2 reversed the anti-osteosarcoma effect of busulfan. (a) U2OS cells were transfected with antisense oligonucleotides (ASO) of miR-200a, miR-200b, miR-200b, miR-141, and miR-429 to downregulate the expression of miRNAs. The percentage of viable cells, apoptosis, β-gal-positive cells and 2'-7'-Dichlorodihydrofluorescein diacetate (DCF-DA) levels were assayed after 24 h. All data were normalized to negative control (NC). (b) MG-63 cells were transfected with pcDNA3.1-ZEB1 or pcDNA3.1-ZEB2, two overexpressing plasmids. The percentage of viable cells, apoptosis, β-gal-positive cells and DCF-DA levels were assayed after 24 h. (c) Downregulation of all five members in the miR-200 family by ASO resulted in upregulation of ZEB1 and ZEB2. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4317920&req=5

fig03: Downregulating the microRNA-200 (miR-200) family or upregulating ZEB1 and ZEB2 reversed the anti-osteosarcoma effect of busulfan. (a) U2OS cells were transfected with antisense oligonucleotides (ASO) of miR-200a, miR-200b, miR-200b, miR-141, and miR-429 to downregulate the expression of miRNAs. The percentage of viable cells, apoptosis, β-gal-positive cells and 2'-7'-Dichlorodihydrofluorescein diacetate (DCF-DA) levels were assayed after 24 h. All data were normalized to negative control (NC). (b) MG-63 cells were transfected with pcDNA3.1-ZEB1 or pcDNA3.1-ZEB2, two overexpressing plasmids. The percentage of viable cells, apoptosis, β-gal-positive cells and DCF-DA levels were assayed after 24 h. (c) Downregulation of all five members in the miR-200 family by ASO resulted in upregulation of ZEB1 and ZEB2. *P < 0.05.
Mentions: To define whether busulfan produced its anti-osteosarcoma effect through downregulation of the miRNA-200 family and by modulation of expression of its downstream targets ZEB1 and ZEB2, we inhibited the expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, or overexpressed ZEB1 and ZEB1, by miRNA antisense oligonucleotide transfection or plasmid transfection (p-ZEB1 or p-ZEB2), respectively. We found that downregulation of all five members in the miR-200 family reversed the effect induced by busulfan in terms of cell viability, proliferation, level of ROS apoptosis, and cell senescence (Fig. 3a). Similarly, we found that upregulation of ZEB1 or ZEB2 expression also reversed the anti-osteosarcoma effect of busulfan (Fig. 3b), without affecting the expression levels of the miR-200 family (data not shown). Of note, downregulation of all five members in the miR-200 family resulted in upregulation of ZEB1 and ZEB2 (Fig. 3c). Taken together, these loss-of-function experiments thus indicate that busulfan may have its anti-osteosarcoma effect by upregulating ZEB1 and ZEB2 through activating the miRNA-200 family.

Bottom Line: Busulfan has been widely used to treat CML.Moreover, a series of loss-of-function and gain-of-function experiments further indicated that busulfan may have its anti-osteosarcoma effect by upregulating the microRNA-200 (miR-200) family which subsequently downregulated its target genes ZEB1 and ZEB2.Taken together, our data suggest that busulfan may have an anti-osteosarcoma effect through downregulating ZEB1 and ZEB2 through activating the miR-200 family, highlighting a possibility of using busulfan as a novel therapy for osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: 169th Hospital, School of Medicine, Hunan Normal University, Changsha, China.

Show MeSH
Related in: MedlinePlus