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Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells.

Venkatesan N, Krishnakumar S, Deepa PR, Deepa M, Khetan V, Reddy MA - Mol. Vis. (2012)

Bottom Line: These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10).Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity.HMGA2 may be considered a promising candidate for gene silencing therapy in RB.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.

ABSTRACT

Aim: To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells.

Methods: Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in post-silenced RB cell lines (Y79 and WERI Rb1). These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10). Zymographic determination of matrix metalloproteinase (MMP) activity was performed in RB cells. A cell cycle assay and a proliferation assay were performed in post-transfected RB cells.

Results: HMGA2 gene silencing in cultured RB cells results in reduced cell proliferation and transition in the G1/S phase. The whole genome microarray analysis of HMGA2 silenced Y79 cells revealed overall upregulation of 1,132 genes (≥ 1.0 fold) and downregulation of 1,562 genes (≤ -1.0 fold). Specific quantitative pathway analysis of the deregulated genes (using Biointerpreter) revealed 150 upregulated genes and 77 downregulated genes (≥ 1.0 fold) involved in vital pathways, namely, mitogen-activated protein kinase, Janus kinase/signal transducers and activators of transcription, Ras pathway, Ras-induced extracellular signal-regulated protein kinases 1 and 2, and tumor protein p53. The differential expression of genes obtained from microarray analysis (Homo sapiens ELK1, member of ETS oncogene family [ELK1], Homo sapiens cyclin-dependent kinase 6 [CDK6], Homo sapiens E2F transcription factor 4, p107/p130-binding [E2F4], Homo sapiens G-2 and S-phase expressed 1 [GTSE1], Damage-regulated autophagy modulator [DRAM], Homo sapiens cadherin 1, type 1,E-cadherin (epithelial) [CDH1], Homo sapiens snail homolog 1 (Drosophila) [SNAI1], Homo sapiens matrix metallopeptidase 2 [MMP2], and Homo sapiens matrix metallopeptidase 9 [MMP9]) was confirmed with quantitative reverse-transcriptase polymerase chain reaction in post-silenced RB cells. Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity.

Conclusions: Our study revealed molecular regulatory changes induced by HMGA2 silencing in RB cancer cells, offering mechanistic insights into the anticancer potential. HMGA2 may be considered a promising candidate for gene silencing therapy in RB.

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Related in: MedlinePlus

The constitutive mRNA expression of selected genes from microarray data in RB primary tumor tissues using real-time quantitative reverse-transcriptase PCR. The error bars represent the standard deviation of triplicate values. Abbreviations: ELK1: member of ETS oncogene family, GTSE1: G-2 and S-phase expressed 1, CDK6: cyclin-dependent kinase 6, E2F4:E2F transcription factor 4, p107/p130-binding, DRAM represents damage-regulated autophagy modulator, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila).
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f8: The constitutive mRNA expression of selected genes from microarray data in RB primary tumor tissues using real-time quantitative reverse-transcriptase PCR. The error bars represent the standard deviation of triplicate values. Abbreviations: ELK1: member of ETS oncogene family, GTSE1: G-2 and S-phase expressed 1, CDK6: cyclin-dependent kinase 6, E2F4:E2F transcription factor 4, p107/p130-binding, DRAM represents damage-regulated autophagy modulator, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila).

Mentions: The expression of the selected panel of genes (ELK1, CDK6, E2F4, GTSE1, DRAM, CDH1, and SNAI1) was compared for their relative expression in non-transfected primary RB tumors. We observed an inverse correlation of gene expression between the untransfected tumors and the HMGA2-silenced RB cells (Figure 8). For the ten RB tumor samples analyzed, the average levels of gene expression as follows: ELK1 (9.21), GTSE1 (6.23), CDK6 (10.76), E2F4 (10.51), DRAM (−4.79), CDH1 (−0.430), and SNAI1 (3.60), Table 2.


Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells.

Venkatesan N, Krishnakumar S, Deepa PR, Deepa M, Khetan V, Reddy MA - Mol. Vis. (2012)

The constitutive mRNA expression of selected genes from microarray data in RB primary tumor tissues using real-time quantitative reverse-transcriptase PCR. The error bars represent the standard deviation of triplicate values. Abbreviations: ELK1: member of ETS oncogene family, GTSE1: G-2 and S-phase expressed 1, CDK6: cyclin-dependent kinase 6, E2F4:E2F transcription factor 4, p107/p130-binding, DRAM represents damage-regulated autophagy modulator, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472926&req=5

f8: The constitutive mRNA expression of selected genes from microarray data in RB primary tumor tissues using real-time quantitative reverse-transcriptase PCR. The error bars represent the standard deviation of triplicate values. Abbreviations: ELK1: member of ETS oncogene family, GTSE1: G-2 and S-phase expressed 1, CDK6: cyclin-dependent kinase 6, E2F4:E2F transcription factor 4, p107/p130-binding, DRAM represents damage-regulated autophagy modulator, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila).
Mentions: The expression of the selected panel of genes (ELK1, CDK6, E2F4, GTSE1, DRAM, CDH1, and SNAI1) was compared for their relative expression in non-transfected primary RB tumors. We observed an inverse correlation of gene expression between the untransfected tumors and the HMGA2-silenced RB cells (Figure 8). For the ten RB tumor samples analyzed, the average levels of gene expression as follows: ELK1 (9.21), GTSE1 (6.23), CDK6 (10.76), E2F4 (10.51), DRAM (−4.79), CDH1 (−0.430), and SNAI1 (3.60), Table 2.

Bottom Line: These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10).Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity.HMGA2 may be considered a promising candidate for gene silencing therapy in RB.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.

ABSTRACT

Aim: To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells.

Methods: Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in post-silenced RB cell lines (Y79 and WERI Rb1). These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10). Zymographic determination of matrix metalloproteinase (MMP) activity was performed in RB cells. A cell cycle assay and a proliferation assay were performed in post-transfected RB cells.

Results: HMGA2 gene silencing in cultured RB cells results in reduced cell proliferation and transition in the G1/S phase. The whole genome microarray analysis of HMGA2 silenced Y79 cells revealed overall upregulation of 1,132 genes (≥ 1.0 fold) and downregulation of 1,562 genes (≤ -1.0 fold). Specific quantitative pathway analysis of the deregulated genes (using Biointerpreter) revealed 150 upregulated genes and 77 downregulated genes (≥ 1.0 fold) involved in vital pathways, namely, mitogen-activated protein kinase, Janus kinase/signal transducers and activators of transcription, Ras pathway, Ras-induced extracellular signal-regulated protein kinases 1 and 2, and tumor protein p53. The differential expression of genes obtained from microarray analysis (Homo sapiens ELK1, member of ETS oncogene family [ELK1], Homo sapiens cyclin-dependent kinase 6 [CDK6], Homo sapiens E2F transcription factor 4, p107/p130-binding [E2F4], Homo sapiens G-2 and S-phase expressed 1 [GTSE1], Damage-regulated autophagy modulator [DRAM], Homo sapiens cadherin 1, type 1,E-cadherin (epithelial) [CDH1], Homo sapiens snail homolog 1 (Drosophila) [SNAI1], Homo sapiens matrix metallopeptidase 2 [MMP2], and Homo sapiens matrix metallopeptidase 9 [MMP9]) was confirmed with quantitative reverse-transcriptase polymerase chain reaction in post-silenced RB cells. Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity.

Conclusions: Our study revealed molecular regulatory changes induced by HMGA2 silencing in RB cancer cells, offering mechanistic insights into the anticancer potential. HMGA2 may be considered a promising candidate for gene silencing therapy in RB.

Show MeSH
Related in: MedlinePlus