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Analysis of Gene Expression Profiles in HeLa Cells in Response to Overexpression or siRNA-mediated Depletion of NASP

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Analysis of Gene Expression Profiles in HeLa Cells in Response to Overexpression or siRNA-mediated Depletion of NASP

Oleg M Alekseev et al. Reprod Biol Endocrinol.

Abstract

Background: NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA.

Methods: Total RNA from HeLa cells overexpressing NASP or depleted of NASP by siRNA treatment was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to whole human genome microarrays (Agilent Technologies, Wilmington, Delaware, USA). Various gene expression analysis techniques were employed: Significance Analysis of Microarrays (SAM), Expression Analysis Systematic Explorer (EASE), and Ingenuity Pathways Analysis (IPA).

Results: From approximately 36 thousand genes present in a total human genome microarray, we identified a set of 47 up-regulated and 7 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 56 up-regulated and 71 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response, and cellular growth and cancer pathways (major "hubs": TNF, FOS, EGR1, NFkappaB, IRF7, STAT1, IL6). Depletion of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major "hubs": E2F8, TP53, FGF, FSH, FST, hCG, NFkappaB, TRAF6).

Conclusion: This study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. We have confirmed the previously reported interactions between NASP and HSP90, HSP70, histone H1, histone H3, and TRAF6. Overexpression and depletion of NASP identified overlapping networks that included TNF as a core protein, confirming that both high and low levels of NASP are detrimental to cell cycle progression. Networks with cancer-related functions had the highest significance, however reproductive networks containing follistatin and FSH were also significantly affected, which confirmed NASP's important role in reproductive tissues. This study revealed that, despite some overlap, each response was associated with a unique gene signature and placed NASP in important cell regulatory networks.

Figures

Figure 1
Figure 1
SAM identification of genes with significant changes in expression. A. Scatter plot of the observed relative difference d(i) versus the expected relative difference dE(i) in cells overexpressing NASP. The solid line indicates the line for d(i) = dE(i), where the observed relative difference is identical to the expected relative difference. The dotted lines are drawn at a distance Delta 11.79 from the solid line. B. Scatter plot of the observed relative difference d(i) versus the expected relative difference dE(i) in cells treated with NASP siRNA. The solid line indicates the line for d(i) = dE(i), where the observed relative difference is identical to the expected relative difference. The dotted lines are drawn at a distance Delta 11.75 from the solid line.
Figure 2
Figure 2
Top functions affected as a result of altered gene expression. Functions determined by: A. Up-regulated genes after NASP overexpression. B. Down-regulated genes after NASP overexpression. C. Up-regulated genes after NASP depletion. D. Down-regulated genes after NASP depletion. Bars represent -log (p-value) for disproportionate representation of affected genes in the total number of genes in the selected function/disease category. Threshold (red line) denotes the p = 0.05 level.
Figure 3
Figure 3
Fragment of molecular network of down-regulated genes in the result of NASP depletion. Shaded shapes present focus genes, clear shapes present interacting genes. Table 6 contains all the genes.
Figure 4
Figure 4
Top canonical pathways affected by. A. Up-regulated genes after NASP overexpression. B. Down-regulated genes after NASP overexpression. C. Up-regulated genes after NASP depletion. D. Down-regulated genes after NASP depletion. Bars represent -log (p-value) for disproportionate representation of affected genes in the selected pathway, yellow line represents the ratio of affected genes to the total number of genes in a pathway. Threshold (red line) denotes the p = 0.05 level.

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