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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 decreases the proliferation of RB (Y79, WERI Rb1) cells. The Y79 cells were treated with high mobility group A2 (HMGA2) short interfering (si)RNA, and the cell proliferation was assessed at 24, 48, and 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell viability decreased to 81.7%, 67.5%, and 45.5% in the Y79 cell line, and to 75.4%, 69.4%, and 49.9% in the WERI Rb1 cell line when compared to Y79 cells that were not treated with siRNA. The error bars represent the standard deviation of triplicate values.
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f4: HMGA2 short interfering RNA treatment decreases the proliferation of RB (Y79, WERI Rb1) cells. The Y79 cells were treated with high mobility group A2 (HMGA2) short interfering (si)RNA, and the cell proliferation was assessed at 24, 48, and 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell viability decreased to 81.7%, 67.5%, and 45.5% in the Y79 cell line, and to 75.4%, 69.4%, and 49.9% in the WERI Rb1 cell line when compared to Y79 cells that were not treated with siRNA. The error bars represent the standard deviation of triplicate values.

Mentions: The effect of HMGA2 gene silencing on modulating cell proliferation was studied using an MTT assay. The MTT assay in the RB (Y79, WERI Rb1) cells treated with the anti-HMGA2 siRNA, scrambled siRNA (a control for non-specific effects of siRNA treatment on cell growth), and the untreated Y79 cells at the end of 24 h, 48 h, and 72 h resulted in a significant decrease in cell proliferation to 81.7%, 67.5%, and 45.5% in Y79 cells and 75.4%, 69.4%, and 49.9% in WERI Rb1 cells, respectively (Figure 4). There was no significant difference in the cell proliferation rate between the scrambled siRNA and untreated Y79 cells.


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 decreases the proliferation of RB (Y79, WERI Rb1) cells. The Y79 cells were treated with high mobility group A2 (HMGA2) short interfering (si)RNA, and the cell proliferation was assessed at 24, 48, and 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell viability decreased to 81.7%, 67.5%, and 45.5% in the Y79 cell line, and to 75.4%, 69.4%, and 49.9% in the WERI Rb1 cell line when compared to Y79 cells that were not treated with siRNA. The error bars represent the standard deviation of triplicate values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: HMGA2 short interfering RNA treatment decreases the proliferation of RB (Y79, WERI Rb1) cells. The Y79 cells were treated with high mobility group A2 (HMGA2) short interfering (si)RNA, and the cell proliferation was assessed at 24, 48, and 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell viability decreased to 81.7%, 67.5%, and 45.5% in the Y79 cell line, and to 75.4%, 69.4%, and 49.9% in the WERI Rb1 cell line when compared to Y79 cells that were not treated with siRNA. The error bars represent the standard deviation of triplicate values.
Mentions: The effect of HMGA2 gene silencing on modulating cell proliferation was studied using an MTT assay. The MTT assay in the RB (Y79, WERI Rb1) cells treated with the anti-HMGA2 siRNA, scrambled siRNA (a control for non-specific effects of siRNA treatment on cell growth), and the untreated Y79 cells at the end of 24 h, 48 h, and 72 h resulted in a significant decrease in cell proliferation to 81.7%, 67.5%, and 45.5% in Y79 cells and 75.4%, 69.4%, and 49.9% in WERI Rb1 cells, respectively (Figure 4). There was no significant difference in the cell proliferation rate between the scrambled siRNA and untreated Y79 cells.

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