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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 leads to changes in the gene expression profile in Y79 cells. A: The heat map represents the expression profile of 100 genes differentially modified in response to knockdown of high mobility group A2 (HMGA2) in Y79 cells compared to untreated cells. The horizontal lines represent the relative fold change in the expression of individual genes modified by the HMGA2-short interfering siRNA. Red and green indicate increased and decreased expression, respectively, relative to non-silenced control Y79 cells. B: Functional grouping of upregulated genes and downregulated genes in Y79 cells treated with HMGA2 short interfering siRNA. The functional grouping of all the distinct gene identification for their known biologic function was done according to gene ontology. C: The interlinking of distinct genes identified for their known biologic function are drawn using the GeneMANIA tool.
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f5: HMGA2 short interfering RNA treatment leads to changes in the gene expression profile in Y79 cells. A: The heat map represents the expression profile of 100 genes differentially modified in response to knockdown of high mobility group A2 (HMGA2) in Y79 cells compared to untreated cells. The horizontal lines represent the relative fold change in the expression of individual genes modified by the HMGA2-short interfering siRNA. Red and green indicate increased and decreased expression, respectively, relative to non-silenced control Y79 cells. B: Functional grouping of upregulated genes and downregulated genes in Y79 cells treated with HMGA2 short interfering siRNA. The functional grouping of all the distinct gene identification for their known biologic function was done according to gene ontology. C: The interlinking of distinct genes identified for their known biologic function are drawn using the GeneMANIA tool.

Mentions: The genome-wide expression of anti-HMGA2 siRNA treated and untreated Y79 cells were analyzed to probe the genes regulated by the HMGA2 gene. As a result of the anti-HMGA2 siRNA treatment in Y79 cells, a total of vital 227 gene transcripts involved in various cellular functions were modulated, which includes 150 upregulated (66.07%) and 77 downregulated (33.9%). Significantly, dysregulated pathways were identified using several databases such as Biologic Pathway Exchange (BioPAX) pathways from Biocarta, Human Protein Reference Database (HPRD), Reactome, KEGG, and NCI-cGAP, by querying the differentially expressed gene list against all the genes annotated with pathway information in the microarray. A total of 100 upregulated and downregulated genes of interest are shown (Figure 5A-C). Gene descriptions for some of the key genes shown are presented in Table 4 and Table 5. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) [37] and are accessible through GEO Series accession number GSE31687.


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 leads to changes in the gene expression profile in Y79 cells. A: The heat map represents the expression profile of 100 genes differentially modified in response to knockdown of high mobility group A2 (HMGA2) in Y79 cells compared to untreated cells. The horizontal lines represent the relative fold change in the expression of individual genes modified by the HMGA2-short interfering siRNA. Red and green indicate increased and decreased expression, respectively, relative to non-silenced control Y79 cells. B: Functional grouping of upregulated genes and downregulated genes in Y79 cells treated with HMGA2 short interfering siRNA. The functional grouping of all the distinct gene identification for their known biologic function was done according to gene ontology. C: The interlinking of distinct genes identified for their known biologic function are drawn using the GeneMANIA tool.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: HMGA2 short interfering RNA treatment leads to changes in the gene expression profile in Y79 cells. A: The heat map represents the expression profile of 100 genes differentially modified in response to knockdown of high mobility group A2 (HMGA2) in Y79 cells compared to untreated cells. The horizontal lines represent the relative fold change in the expression of individual genes modified by the HMGA2-short interfering siRNA. Red and green indicate increased and decreased expression, respectively, relative to non-silenced control Y79 cells. B: Functional grouping of upregulated genes and downregulated genes in Y79 cells treated with HMGA2 short interfering siRNA. The functional grouping of all the distinct gene identification for their known biologic function was done according to gene ontology. C: The interlinking of distinct genes identified for their known biologic function are drawn using the GeneMANIA tool.
Mentions: The genome-wide expression of anti-HMGA2 siRNA treated and untreated Y79 cells were analyzed to probe the genes regulated by the HMGA2 gene. As a result of the anti-HMGA2 siRNA treatment in Y79 cells, a total of vital 227 gene transcripts involved in various cellular functions were modulated, which includes 150 upregulated (66.07%) and 77 downregulated (33.9%). Significantly, dysregulated pathways were identified using several databases such as Biologic Pathway Exchange (BioPAX) pathways from Biocarta, Human Protein Reference Database (HPRD), Reactome, KEGG, and NCI-cGAP, by querying the differentially expressed gene list against all the genes annotated with pathway information in the microarray. A total of 100 upregulated and downregulated genes of interest are shown (Figure 5A-C). Gene descriptions for some of the key genes shown are presented in Table 4 and Table 5. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) [37] and are accessible through GEO Series accession number GSE31687.

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