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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

Effect of small interfering RNA on the expression of HMGA2 in RB Y79 cells in vitro. A: The mRNA levels of high mobility group A2 (HMGA2) in RB cells (Y79) treated with HMGA2 short interfering (si) RNA, namely, the siRNA.1 (Hs_HMGA2_6) sequence, led to a 4.65 log2 ratio decrease, siRNA.2 (Hs_HMGA2_7) led to a decrease of 2.0, the siRNA.3 sequence led to an increase of 1.76 log2 ratio when compared with HMGA2 mRNA levels in control (solid bar) and to a decrease of a 1.68 log2 ratio, 4.175 log2 ratio, an increase of a 2.65 log2 ratio, respectively, to cells treated with scrambled (SCR) siRNA (dotted bars) at the end of 48 h. The error bars represent the standard deviation of triplicate values. B: The mRNA levels of high mobility group A2 (HMGA2) in Y79 cells treated with HMGA2 short interfering (si) RNA [siRNA.1 (Hs_HMGA2_6) sequence] led to a decrease of a 4.65 log2 ratio, in WERI Rb1 led to a decrease of a 2.56 log2 ratio when compared with HMGA2 mRNA levels in RB control cells (solid bar) and to a decrease of a 4.175 log2 ratio, a 3.17 log2 ratio, respectively, in RB (Y79, WERI Rb1) cells treated with scrambled (SCR) siRNA (dotted bars at the end of 48 h). The error bars represent the standard deviation of triplicate values.
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f1: Effect of small interfering RNA on the expression of HMGA2 in RB Y79 cells in vitro. A: The mRNA levels of high mobility group A2 (HMGA2) in RB cells (Y79) treated with HMGA2 short interfering (si) RNA, namely, the siRNA.1 (Hs_HMGA2_6) sequence, led to a 4.65 log2 ratio decrease, siRNA.2 (Hs_HMGA2_7) led to a decrease of 2.0, the siRNA.3 sequence led to an increase of 1.76 log2 ratio when compared with HMGA2 mRNA levels in control (solid bar) and to a decrease of a 1.68 log2 ratio, 4.175 log2 ratio, an increase of a 2.65 log2 ratio, respectively, to cells treated with scrambled (SCR) siRNA (dotted bars) at the end of 48 h. The error bars represent the standard deviation of triplicate values. B: The mRNA levels of high mobility group A2 (HMGA2) in Y79 cells treated with HMGA2 short interfering (si) RNA [siRNA.1 (Hs_HMGA2_6) sequence] led to a decrease of a 4.65 log2 ratio, in WERI Rb1 led to a decrease of a 2.56 log2 ratio when compared with HMGA2 mRNA levels in RB control cells (solid bar) and to a decrease of a 4.175 log2 ratio, a 3.17 log2 ratio, respectively, in RB (Y79, WERI Rb1) cells treated with scrambled (SCR) siRNA (dotted bars at the end of 48 h). The error bars represent the standard deviation of triplicate values.

Mentions: Initially, the HMGA2 gene silencing protocol was optimized in cultured RB cells. Using qRT–PCR analyses, we found that over a period of 48 h, transfection with the siRNA.1 (Hs_HMGA2_6) sequence led to a −4.65 log2 ratio decrease, while the other sequences, siRNA.2 (Hs_HMGA2_7) and siRNA.3 sequence, led to a log2 ratio of −2.0 and 1.76 decrease, respectively, only when compared with and without scrambled siRNA as controls in RB cells (Y79; Figure 1A). This result when using the siRNA.1 (Hs_HMGA2_6) sequence in the study is consistent with the western blot analysis of the same in RB cells (Y79; Figure 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)

Effect of small interfering RNA on the expression of HMGA2 in RB Y79 cells in vitro. A: The mRNA levels of high mobility group A2 (HMGA2) in RB cells (Y79) treated with HMGA2 short interfering (si) RNA, namely, the siRNA.1 (Hs_HMGA2_6) sequence, led to a 4.65 log2 ratio decrease, siRNA.2 (Hs_HMGA2_7) led to a decrease of 2.0, the siRNA.3 sequence led to an increase of 1.76 log2 ratio when compared with HMGA2 mRNA levels in control (solid bar) and to a decrease of a 1.68 log2 ratio, 4.175 log2 ratio, an increase of a 2.65 log2 ratio, respectively, to cells treated with scrambled (SCR) siRNA (dotted bars) at the end of 48 h. The error bars represent the standard deviation of triplicate values. B: The mRNA levels of high mobility group A2 (HMGA2) in Y79 cells treated with HMGA2 short interfering (si) RNA [siRNA.1 (Hs_HMGA2_6) sequence] led to a decrease of a 4.65 log2 ratio, in WERI Rb1 led to a decrease of a 2.56 log2 ratio when compared with HMGA2 mRNA levels in RB control cells (solid bar) and to a decrease of a 4.175 log2 ratio, a 3.17 log2 ratio, respectively, in RB (Y79, WERI Rb1) cells treated with scrambled (SCR) siRNA (dotted bars at the end of 48 h). The error bars represent the standard deviation of triplicate values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Effect of small interfering RNA on the expression of HMGA2 in RB Y79 cells in vitro. A: The mRNA levels of high mobility group A2 (HMGA2) in RB cells (Y79) treated with HMGA2 short interfering (si) RNA, namely, the siRNA.1 (Hs_HMGA2_6) sequence, led to a 4.65 log2 ratio decrease, siRNA.2 (Hs_HMGA2_7) led to a decrease of 2.0, the siRNA.3 sequence led to an increase of 1.76 log2 ratio when compared with HMGA2 mRNA levels in control (solid bar) and to a decrease of a 1.68 log2 ratio, 4.175 log2 ratio, an increase of a 2.65 log2 ratio, respectively, to cells treated with scrambled (SCR) siRNA (dotted bars) at the end of 48 h. The error bars represent the standard deviation of triplicate values. B: The mRNA levels of high mobility group A2 (HMGA2) in Y79 cells treated with HMGA2 short interfering (si) RNA [siRNA.1 (Hs_HMGA2_6) sequence] led to a decrease of a 4.65 log2 ratio, in WERI Rb1 led to a decrease of a 2.56 log2 ratio when compared with HMGA2 mRNA levels in RB control cells (solid bar) and to a decrease of a 4.175 log2 ratio, a 3.17 log2 ratio, respectively, in RB (Y79, WERI Rb1) cells treated with scrambled (SCR) siRNA (dotted bars at the end of 48 h). The error bars represent the standard deviation of triplicate values.
Mentions: Initially, the HMGA2 gene silencing protocol was optimized in cultured RB cells. Using qRT–PCR analyses, we found that over a period of 48 h, transfection with the siRNA.1 (Hs_HMGA2_6) sequence led to a −4.65 log2 ratio decrease, while the other sequences, siRNA.2 (Hs_HMGA2_7) and siRNA.3 sequence, led to a log2 ratio of −2.0 and 1.76 decrease, respectively, only when compared with and without scrambled siRNA as controls in RB cells (Y79; Figure 1A). This result when using the siRNA.1 (Hs_HMGA2_6) sequence in the study is consistent with the western blot analysis of the same in RB cells (Y79; Figure 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