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. 2013 Sep 18:7:289-306.
doi: 10.4137/BBI.S12494. eCollection 2013.

Molecular Insights on Post-chemotherapy Retinoblastoma by Microarray Gene Expression Analysis

Venkatesan Nalini  1 , Ramya Segu, Perinkulam Ravi Deepa, Vikas Khetan, Madavan Vasudevan, Subramanian Krishnakumar
Affiliations

Molecular Insights on Post-chemotherapy Retinoblastoma by Microarray Gene Expression Analysis

Venkatesan Nalini et al. Bioinform Biol Insights. .

Abstract

Purpose: Management of Retinoblastoma (RB), a pediatric ocular cancer is limited by drug-resistance and drug-dosage related side effects during chemotherapy. Molecular de-regulation in post-chemotherapy RB tumors was investigated.

Materials and methods: cDNA microarray analysis of two post-chemotherapy and one pre-chemotherapy RB tumor tissues was performed, followed by Principle Component Analysis, Gene ontology, Pathway Enrichment analysis and Biological Analysis Network (BAN) modeling. The drug modulation role of two significantly up-regulated genes (p≤0.05) - Ect2 (Epithelial-cell-transforming-sequence-2), and PRAME (preferentially-expressed-Antigen-in-Melanoma) was assessed by qRT-PCR, immunohistochemistry and cell viability assays.

Results: Differential up-regulation of 1672 genes and down-regulation of 2538 genes was observed in RB tissues (relative to normal adult retina), while 1419 genes were commonly de-regulated between pre-chemotherapy and post- chemotherapy RB. Twenty one key gene ontology categories, pathways, biomarkers and phenotype groups harboring 250 differentially expressed genes were dys-regulated (EZH2, NCoR1, MYBL2, RB1, STAMN1, SYK, JAK1/2, STAT1/2, PLK2/4, BIRC5, LAMN1, Ect2, PRAME and ABCC4). Differential molecular expressions of PRAME and Ect2 in RB tumors with and without chemotherapy were analyzed. There was neither up- regulation of MRP1, nor any significant shift in chemotherapeutic IC50, in PRAME over-expressed versus non-transfected RB cells.

Conclusion: Cell cycle regulatory genes were dys-regulated post-chemotherapy. Ect2 gene was expressed in response to chemotherapy-induced stress. PRAME does not contribute to drug resistance in RB, yet its nuclear localization and BAN information, points to its possible regulatory role in RB.

Keywords: Ect2; MYBL2; NCoR1; PRAME; RB; chemotherapy; drug resistance; micro array.

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Figures

Figure 1
Figure 1
The principal component analysis (PCA) of normal retina, RB tumor (pre-chemotherapy RB tumor tissue) and post-chemotherapy RB tumor tissues (RB1 and RB2). The PCA shows clustering of the gene profiles in groups respectively.
Figure 2
Figure 2
(A) The volcano plot showing differentially expressed 4210 genes (includes 1672 upregulated genes and 2538 downregulated genes) in number identified in the gene expression profiling of RB tumors compared with normal retina. (B) The volcano plot showing differentially expressed 1832 genes (includes 1011 upregulated genes and 821 downregulated genes) in number identified in the gene expression profiling of post-chemotherapy RB tumors compared with prechemotherapy RB. (C) Venn diagram representing distribution of differentially expressed genes upon chemo treatment and in tumor sample compared with normal retina. (D) Key biological categories and pathways that were dysregulated in the RB tumors. (E) The heat map represents the expression profile of 15 differentially expressed genes in RB (pre-chemotherapy treated RB, post-chemotherapy treated RB1 and post-chemotherapy treated RB2) compared with normal retina. The horizontal lines represent the relative fold change in the expression of individual genes. Green and red indicate increased and decreased gene expression, respectively, while yellow represents normal expression.
Figure 2
Figure 2
(A) The volcano plot showing differentially expressed 4210 genes (includes 1672 upregulated genes and 2538 downregulated genes) in number identified in the gene expression profiling of RB tumors compared with normal retina. (B) The volcano plot showing differentially expressed 1832 genes (includes 1011 upregulated genes and 821 downregulated genes) in number identified in the gene expression profiling of post-chemotherapy RB tumors compared with prechemotherapy RB. (C) Venn diagram representing distribution of differentially expressed genes upon chemo treatment and in tumor sample compared with normal retina. (D) Key biological categories and pathways that were dysregulated in the RB tumors. (E) The heat map represents the expression profile of 15 differentially expressed genes in RB (pre-chemotherapy treated RB, post-chemotherapy treated RB1 and post-chemotherapy treated RB2) compared with normal retina. The horizontal lines represent the relative fold change in the expression of individual genes. Green and red indicate increased and decreased gene expression, respectively, while yellow represents normal expression.
Figure 3
Figure 3
Protein interaction and regulatory network showing genes and biological process clustering (obtained using cytoscape V 8.0) underlying chemo treatment change versus tumor profile (A) post-chemotherapy and (B) pre-chemotherapy treated RB tumors.
Figure 4
Figure 4
Immunohistostaining of 3 representative RB tumor tissues compared with nonneoplastic retina (DAB staining with hematoxylin counter staining). (A) The photomicrograph shows the strong nuclear expression of PRAME (arrows show positivity) in a RB tumor (40× magnification). (B) The photomicrograph shows the strong nuclear expression of PRAME (arrows show positivity) in a RB tumor (20× magnification). (C) The photomicrograph shows lesser percentage of nuclear of PRAME (arrows show positivity) in a RB tumor (40× magnification). (D) The photomicrograph shows the negative expression of PRAME (arrows show negativity) in the retinal layers (40× magnification).
Figure 5
Figure 5
The mRNA expression of PRAME (grey bar) and Ect2 (black bar) analyzed by real time quantitative reverse transcriptase PCR (qRT-PCR). Values are expressed as mean ± SD of triplicate analyses. *Indicates the post-chemotherapy RB tumor tissues.
Figure 6
Figure 6
qRT-PCR analysis of the relative mRNA expression of PRAME and MRP1 gene in PRAME vector transfected Y79 (retinoblastoma cell line) normalized with untransfected Y79 cells.
Figure 7
Figure 7
Schematic representation of Ect2 mRNA levels in retinoblastoma tumor tissues (green spheres represent post-chemotherapy RB tumor tissues, red spheres represent prechemotherapy RB tissues, and the blue line indicates fold change ± 2.0).
Figure 8
Figure 8
(A) IC50 determination of vincristine in PRAME over expressed and control RB (Y79) cells. (B) IC50 determination of etoposide in PRAME overexpressed and control RB (Y79) cells. (C) IC50 determination of carboplatin in PRAME overexpressed and control RB (Y79) cells.
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