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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 mRNA expression of selected genes from the microarray data was confirmed using real-time quantitative reverse-transcriptase PCR. The black bars represent the mRNA levels quantified with qRT–PCR in the HMGA2-short interfering siRNA treated Y79 cells, and the spotted bars represent the fold expression of genes in the HMGA2-short interfering (siRNA) treated WERI Rb1 cells. The error bars represent the standard deviation of triplicate values. Abbreviations: DRAM represents damage-regulated autophagy modulator, 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, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila), MMP2: matrix metallopeptidase 2, MMP 9: matrix metallopeptidase 9.
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f7: The mRNA expression of selected genes from the microarray data was confirmed using real-time quantitative reverse-transcriptase PCR. The black bars represent the mRNA levels quantified with qRT–PCR in the HMGA2-short interfering siRNA treated Y79 cells, and the spotted bars represent the fold expression of genes in the HMGA2-short interfering (siRNA) treated WERI Rb1 cells. The error bars represent the standard deviation of triplicate values. Abbreviations: DRAM represents damage-regulated autophagy modulator, 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, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila), MMP2: matrix metallopeptidase 2, MMP 9: matrix metallopeptidase 9.

Mentions: The gene expression level of nine genes (ELK1, CDK6, E2F4, GTSE1, DRAM, CDH1, SNAI1, MMP2, and MMP9) in the microarray analysis was consistent with the qRT–PCR findings in the transfected Y79 cells. Although most of the genes were consistent in the expression obtained with the microarray and qRT–PCR analyses, a few genes in the post-transfected WERI Rb1 cells differed in levels of expression with respect to microarray findings. These genes include ELK1, CDK6, and E2F4, which were not downregulated, unlike in the Y79 cells. The SNAI1 gene that was significantly downregulated in Y79 cells was not downregulated to the same extent in the HMGA2-silenced WERI Rb1 cells (that is, the expression level was not below the −1.0 cutoff value; Figure 7).


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 mRNA expression of selected genes from the microarray data was confirmed using real-time quantitative reverse-transcriptase PCR. The black bars represent the mRNA levels quantified with qRT–PCR in the HMGA2-short interfering siRNA treated Y79 cells, and the spotted bars represent the fold expression of genes in the HMGA2-short interfering (siRNA) treated WERI Rb1 cells. The error bars represent the standard deviation of triplicate values. Abbreviations: DRAM represents damage-regulated autophagy modulator, 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, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila), MMP2: matrix metallopeptidase 2, MMP 9: matrix metallopeptidase 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: The mRNA expression of selected genes from the microarray data was confirmed using real-time quantitative reverse-transcriptase PCR. The black bars represent the mRNA levels quantified with qRT–PCR in the HMGA2-short interfering siRNA treated Y79 cells, and the spotted bars represent the fold expression of genes in the HMGA2-short interfering (siRNA) treated WERI Rb1 cells. The error bars represent the standard deviation of triplicate values. Abbreviations: DRAM represents damage-regulated autophagy modulator, 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, CDH1: cadherin 1, type 1, E-cadherin (epithelial; 1), SNAI1: snail homolog 1 (Drosophila), MMP2: matrix metallopeptidase 2, MMP 9: matrix metallopeptidase 9.
Mentions: The gene expression level of nine genes (ELK1, CDK6, E2F4, GTSE1, DRAM, CDH1, SNAI1, MMP2, and MMP9) in the microarray analysis was consistent with the qRT–PCR findings in the transfected Y79 cells. Although most of the genes were consistent in the expression obtained with the microarray and qRT–PCR analyses, a few genes in the post-transfected WERI Rb1 cells differed in levels of expression with respect to microarray findings. These genes include ELK1, CDK6, and E2F4, which were not downregulated, unlike in the Y79 cells. The SNAI1 gene that was significantly downregulated in Y79 cells was not downregulated to the same extent in the HMGA2-silenced WERI Rb1 cells (that is, the expression level was not below the −1.0 cutoff value; Figure 7).

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