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The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

Yadavilli S, Scafidi J, Becher OJ, Saratsis AM, Hiner RL, Kambhampati M, Mariarita S, MacDonald TJ, Codispoti KE, Magge SN, Jaiswal JK, Packer RJ, Nazarian J - Oncotarget (2015)

Bottom Line: We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2.NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors.This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Genetic Medicine, Children's National Health System, Washington, DC, USA.

ABSTRACT
Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

No MeSH data available.


Related in: MedlinePlus

NG2 upregulation in DIPG is partially due to hypermethylation and downregulation of its regulatory microRNA, miR129-2(a) Luciferase vector with 3′-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc-3′UTR) and plasmid vector with miR129-2 sequence were used. PDGFB mouse neurospheres were transfected in triplicates with luciferase and miR129-2 vectors, as shown, and luciferase expression was determined. Co-expression of Luc-3′UTR of NG2 and miR129-2 resulted in significant downregulation of luciferase expression as compared to cells transfected with luc-3′UTR and empty vector. P < 0.05 was considered significant. (b) PDGFB mouse neurospheres were transfected with miR129-2 or NG2-shRNA plasmids. Upregulation of either miR129-2 or NG2-shRNA resulted in NG2 downregulation in vitro when compared to empty vector or control shRNA treated cells. (c) Human DIPG cells, SF8628, were transduced with GFP expressing pCDH-control or pCDH-miR129-2 lentivirus. Immunofluorescence analysis by confocal microscopy revealed upregulation of miR129-2 (indicated by GFP) resulted in NG2 (red) downregulation in vitro when compared to control transduced cells. DAPI was used to stain nuclei. Scale bar = 5 μm. (d) Average expression of miR129-2 as assessed by RT-PCR (*miR129) or illumina expression chip assay (miR129), miR129 hypermethylation (miR129 me), and NG2 mRNA levels in human DIPG compared to adjacent normal. miR129-2 is shown to be downregulated in DIPG as assessed by RT-PCR and illumina chip assays. Hypermethylation of miR129-2 at four or more CpG sites corresponding to miR129-2 promoter were detected in 57% of DIPGs. As expected, downregulation of miR129-2 due to hypermethylation resulted in upregulation of NG2 mRNA as detected by NG2 mRNA using illumina platform. (e) To reverse miR129-2 hypermethylation, PDGFB mouse neurospheres were treated with 5-Azacytidine, a DNA methyl-transferase inhibitor drug. NG2 expression decreased in mouse tumor cells when treated with 10-30 μM 5-Azacytidine as compared to untreated cells. NG2 expression in each sample was normalized to corresponding GAPDH signal and average NG2 expression from three experiments was plotted.
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Figure 3: NG2 upregulation in DIPG is partially due to hypermethylation and downregulation of its regulatory microRNA, miR129-2(a) Luciferase vector with 3′-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc-3′UTR) and plasmid vector with miR129-2 sequence were used. PDGFB mouse neurospheres were transfected in triplicates with luciferase and miR129-2 vectors, as shown, and luciferase expression was determined. Co-expression of Luc-3′UTR of NG2 and miR129-2 resulted in significant downregulation of luciferase expression as compared to cells transfected with luc-3′UTR and empty vector. P < 0.05 was considered significant. (b) PDGFB mouse neurospheres were transfected with miR129-2 or NG2-shRNA plasmids. Upregulation of either miR129-2 or NG2-shRNA resulted in NG2 downregulation in vitro when compared to empty vector or control shRNA treated cells. (c) Human DIPG cells, SF8628, were transduced with GFP expressing pCDH-control or pCDH-miR129-2 lentivirus. Immunofluorescence analysis by confocal microscopy revealed upregulation of miR129-2 (indicated by GFP) resulted in NG2 (red) downregulation in vitro when compared to control transduced cells. DAPI was used to stain nuclei. Scale bar = 5 μm. (d) Average expression of miR129-2 as assessed by RT-PCR (*miR129) or illumina expression chip assay (miR129), miR129 hypermethylation (miR129 me), and NG2 mRNA levels in human DIPG compared to adjacent normal. miR129-2 is shown to be downregulated in DIPG as assessed by RT-PCR and illumina chip assays. Hypermethylation of miR129-2 at four or more CpG sites corresponding to miR129-2 promoter were detected in 57% of DIPGs. As expected, downregulation of miR129-2 due to hypermethylation resulted in upregulation of NG2 mRNA as detected by NG2 mRNA using illumina platform. (e) To reverse miR129-2 hypermethylation, PDGFB mouse neurospheres were treated with 5-Azacytidine, a DNA methyl-transferase inhibitor drug. NG2 expression decreased in mouse tumor cells when treated with 10-30 μM 5-Azacytidine as compared to untreated cells. NG2 expression in each sample was normalized to corresponding GAPDH signal and average NG2 expression from three experiments was plotted.

Mentions: To investigate potential molecular mechanism of NG2 regulation, we used Ingenuity Pathway Analysis (IPA) software, which generates biologic pathways based on published molecular interactions [8]. IPA analysis identified miR129-2 as a potential NG2 regulator. To validate NG2 as a target for miR129-2, we transfected PDGFB mouse neurospheres with a vector containing the 3′-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc-3′UTR) along with a plasmid vector containing miR129-2. We found that co-expression of Luc-3′UTR and miR129-2 results in significant downregulation of luciferase expression as compared to cells transfected with luc-3′UTR vector alone (Figure 3a). Moreover, transient transfection of PDGFB mouse tumor cells with NG2-targeting shRNA or miR129-2 expressing plasmid resulted in downregulation [(76% and 63%, respectively) as compared to control shRNA treated or empty vector treated cells] of NG2 protein as assessed by Western blot assay, validating the role of miR129-2 in regulation of NG2 expression (Figure 3b).


The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

Yadavilli S, Scafidi J, Becher OJ, Saratsis AM, Hiner RL, Kambhampati M, Mariarita S, MacDonald TJ, Codispoti KE, Magge SN, Jaiswal JK, Packer RJ, Nazarian J - Oncotarget (2015)

NG2 upregulation in DIPG is partially due to hypermethylation and downregulation of its regulatory microRNA, miR129-2(a) Luciferase vector with 3′-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc-3′UTR) and plasmid vector with miR129-2 sequence were used. PDGFB mouse neurospheres were transfected in triplicates with luciferase and miR129-2 vectors, as shown, and luciferase expression was determined. Co-expression of Luc-3′UTR of NG2 and miR129-2 resulted in significant downregulation of luciferase expression as compared to cells transfected with luc-3′UTR and empty vector. P < 0.05 was considered significant. (b) PDGFB mouse neurospheres were transfected with miR129-2 or NG2-shRNA plasmids. Upregulation of either miR129-2 or NG2-shRNA resulted in NG2 downregulation in vitro when compared to empty vector or control shRNA treated cells. (c) Human DIPG cells, SF8628, were transduced with GFP expressing pCDH-control or pCDH-miR129-2 lentivirus. Immunofluorescence analysis by confocal microscopy revealed upregulation of miR129-2 (indicated by GFP) resulted in NG2 (red) downregulation in vitro when compared to control transduced cells. DAPI was used to stain nuclei. Scale bar = 5 μm. (d) Average expression of miR129-2 as assessed by RT-PCR (*miR129) or illumina expression chip assay (miR129), miR129 hypermethylation (miR129 me), and NG2 mRNA levels in human DIPG compared to adjacent normal. miR129-2 is shown to be downregulated in DIPG as assessed by RT-PCR and illumina chip assays. Hypermethylation of miR129-2 at four or more CpG sites corresponding to miR129-2 promoter were detected in 57% of DIPGs. As expected, downregulation of miR129-2 due to hypermethylation resulted in upregulation of NG2 mRNA as detected by NG2 mRNA using illumina platform. (e) To reverse miR129-2 hypermethylation, PDGFB mouse neurospheres were treated with 5-Azacytidine, a DNA methyl-transferase inhibitor drug. NG2 expression decreased in mouse tumor cells when treated with 10-30 μM 5-Azacytidine as compared to untreated cells. NG2 expression in each sample was normalized to corresponding GAPDH signal and average NG2 expression from three experiments was plotted.
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Figure 3: NG2 upregulation in DIPG is partially due to hypermethylation and downregulation of its regulatory microRNA, miR129-2(a) Luciferase vector with 3′-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc-3′UTR) and plasmid vector with miR129-2 sequence were used. PDGFB mouse neurospheres were transfected in triplicates with luciferase and miR129-2 vectors, as shown, and luciferase expression was determined. Co-expression of Luc-3′UTR of NG2 and miR129-2 resulted in significant downregulation of luciferase expression as compared to cells transfected with luc-3′UTR and empty vector. P < 0.05 was considered significant. (b) PDGFB mouse neurospheres were transfected with miR129-2 or NG2-shRNA plasmids. Upregulation of either miR129-2 or NG2-shRNA resulted in NG2 downregulation in vitro when compared to empty vector or control shRNA treated cells. (c) Human DIPG cells, SF8628, were transduced with GFP expressing pCDH-control or pCDH-miR129-2 lentivirus. Immunofluorescence analysis by confocal microscopy revealed upregulation of miR129-2 (indicated by GFP) resulted in NG2 (red) downregulation in vitro when compared to control transduced cells. DAPI was used to stain nuclei. Scale bar = 5 μm. (d) Average expression of miR129-2 as assessed by RT-PCR (*miR129) or illumina expression chip assay (miR129), miR129 hypermethylation (miR129 me), and NG2 mRNA levels in human DIPG compared to adjacent normal. miR129-2 is shown to be downregulated in DIPG as assessed by RT-PCR and illumina chip assays. Hypermethylation of miR129-2 at four or more CpG sites corresponding to miR129-2 promoter were detected in 57% of DIPGs. As expected, downregulation of miR129-2 due to hypermethylation resulted in upregulation of NG2 mRNA as detected by NG2 mRNA using illumina platform. (e) To reverse miR129-2 hypermethylation, PDGFB mouse neurospheres were treated with 5-Azacytidine, a DNA methyl-transferase inhibitor drug. NG2 expression decreased in mouse tumor cells when treated with 10-30 μM 5-Azacytidine as compared to untreated cells. NG2 expression in each sample was normalized to corresponding GAPDH signal and average NG2 expression from three experiments was plotted.
Mentions: To investigate potential molecular mechanism of NG2 regulation, we used Ingenuity Pathway Analysis (IPA) software, which generates biologic pathways based on published molecular interactions [8]. IPA analysis identified miR129-2 as a potential NG2 regulator. To validate NG2 as a target for miR129-2, we transfected PDGFB mouse neurospheres with a vector containing the 3′-UTR sequence of NG2 cloned downstream of the firefly luciferase gene (Luc-3′UTR) along with a plasmid vector containing miR129-2. We found that co-expression of Luc-3′UTR and miR129-2 results in significant downregulation of luciferase expression as compared to cells transfected with luc-3′UTR vector alone (Figure 3a). Moreover, transient transfection of PDGFB mouse tumor cells with NG2-targeting shRNA or miR129-2 expressing plasmid resulted in downregulation [(76% and 63%, respectively) as compared to control shRNA treated or empty vector treated cells] of NG2 protein as assessed by Western blot assay, validating the role of miR129-2 in regulation of NG2 expression (Figure 3b).

Bottom Line: We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2.NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors.This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Genetic Medicine, Children's National Health System, Washington, DC, USA.

ABSTRACT
Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

No MeSH data available.


Related in: MedlinePlus