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 inhibited by siRNA.
Methods
Total RNA was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to oligonucleotide microarrays.
Results
From approximately 36 thousand genes present in a total human genome microarray, we identified a set of 57 up-regulated and 11 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 68 up-regulated and 92 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, NFkB, IRF7, STAT1, IL6). Inhibition 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, NFkB, TRAF6).
Conclusions
This study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. Most of these genes have not been previously reported to be connected to expression levels of NASP. Gene ontology and protein network analysis identified general biological processes as well as individual genes/gene products and possible interaction networks. Networks with cancer-related functions had the highest significance. We found signaling pathways which were affected as a result of changed NASP expression. This analysis revealed that, despite some overlap, each reactive response was associated with a unique gene signature. These results place NASP in important cell regulatory networks and suggest a number of directions for further research.
Overall design: Total cellular RNA was purified from HeLa cells using RNeasy ® Mini Kit (Qiagen, Valencia, CA) according to manufacturers’ instructions. RNA samples representing four separate experiments from cells overexpressing tNASP and four experiments from cells treated with NASP siRNA, along with appropriate controls, were submitted for analysis. After the RNA Quality check was performed the samples of RNA from treated cells were amplified with incorporation of Cy5-CTP (fluorescent in the red region), while control samples were labeled by Cy3-CTP (fluorescent in the green region) and purified. The labeled cRNA samples were then fragmented in fragmentation buffer at 60°C for 30 min before the microarray hybridization. Each sample was hybridized to a whole separate Human Genome (4×44K) microarray (Agilent Technologies, Wilmington, DE) overnight at 65°C in a hybridization oven. The hybridization slides were washed, stabilized, dried, and immediately scanned by Agilent Technologies Microarray Scanner (Agilent Technologies, Wilmington, DE). RNA hybridization was performed in the Genomics Core and Microarray Facility (Lineberger Comprehensive Cancer Center, UNC-CH) according to the protocol suggested by Agilent (Agilent Technologies, Wilmington, DE).
Statistical analysis – During the initial analysis at UNC Microarray Database, all genes were retrieved, appropriately annotated, and filtered. Eventually, only genes with an absolute value of a Log2 Red/Green Lowess Normalized Ratio of at least 1 (doubled in intensity) for all 4 arrays were selected.
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