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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

HMGA2 short interfering RNA treatment results in the marked arrest of the cell cycle phase. A: Untreated Y79 cells. B: Scrambled siRNA treated Y79 cells. C: HMGA2 siRNA treated Y79 cells showing marked G:0/G:1 and S: phase cell cycle arrest compared to the control cells. D: Untreated WERI Rb1 cells. E: Scrambled siRNA treated WERI Rb1 cells. F: HMGA2 siRNA-treated WERI Rb1 cells show marked G:2/M phase cell cycle arrest compared to the control cells.
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f3: HMGA2 short interfering RNA treatment results in the marked arrest of the cell cycle phase. A: Untreated Y79 cells. B: Scrambled siRNA treated Y79 cells. C: HMGA2 siRNA treated Y79 cells showing marked G:0/G:1 and S: phase cell cycle arrest compared to the control cells. D: Untreated WERI Rb1 cells. E: Scrambled siRNA treated WERI Rb1 cells. F: HMGA2 siRNA-treated WERI Rb1 cells show marked G:2/M phase cell cycle arrest compared to the control cells.

Mentions: The effect of HMGA2 gene silencing on modulating RB cell growth was studied using a cell cycle assay after transfection with HMGA2 siRNA (Table 3). Cell cycle distribution was assessed with flow cytometry. Compared to the untreated cells, the G0/G1 and S phases in the post-transfected Y79 cells, and the G0/G1, S, and G2/M phases in post-transfected WERI Rb1 cells showed marked cell cycle arrest (Figure 3).


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)

HMGA2 short interfering RNA treatment results in the marked arrest of the cell cycle phase. A: Untreated Y79 cells. B: Scrambled siRNA treated Y79 cells. C: HMGA2 siRNA treated Y79 cells showing marked G:0/G:1 and S: phase cell cycle arrest compared to the control cells. D: Untreated WERI Rb1 cells. E: Scrambled siRNA treated WERI Rb1 cells. F: HMGA2 siRNA-treated WERI Rb1 cells show marked G:2/M phase cell cycle arrest compared to the control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: HMGA2 short interfering RNA treatment results in the marked arrest of the cell cycle phase. A: Untreated Y79 cells. B: Scrambled siRNA treated Y79 cells. C: HMGA2 siRNA treated Y79 cells showing marked G:0/G:1 and S: phase cell cycle arrest compared to the control cells. D: Untreated WERI Rb1 cells. E: Scrambled siRNA treated WERI Rb1 cells. F: HMGA2 siRNA-treated WERI Rb1 cells show marked G:2/M phase cell cycle arrest compared to the control cells.
Mentions: The effect of HMGA2 gene silencing on modulating RB cell growth was studied using a cell cycle assay after transfection with HMGA2 siRNA (Table 3). Cell cycle distribution was assessed with flow cytometry. Compared to the untreated cells, the G0/G1 and S phases in the post-transfected Y79 cells, and the G0/G1, S, and G2/M phases in post-transfected WERI Rb1 cells showed marked cell cycle arrest (Figure 3).

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