Effect of PTTG on endogenous gene expression in HEK 293 cells

Abstract

Background: Pituitary tumor transforming gene (PTTG), also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293) cells that do not express PTTG and analyzed the downstream genes using microarray analysis.

Results: A total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA) and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad) or adenovirus vector expressing GFP (AdGFP). Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of < or = 0.05 and a fold change of > or =2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family.

Conclusion: PTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study demonstrated that PTTG may induce apoptosis by down-regulation of oncogenes such as v-Jun and v-maf and up-regulation of the histone family of genes.

Figure 1

Expression of AdGFP. HEK293 cells were infected with AdGFP protein after 24 h post-infection.

Figure 2

Western blot analysis of PTTG protein expression in HEK293 cells infected with empty adenovirus, adenovirus with GFP and adenovirus with PTTG cDNA.

Figure 3

PTTG down-regulated Jun and Maf expression. A: Normalized expression of v-Jun and v-Maf in negative control, GFP control, and PTTG-overexpressed HEK293 cells by microarray analysis. The normalized expression values are mean ± SD (n = 3). The numbers above the bar represent the fold change of treatment with control group.

Figure 4

PTTG down-regulated Jun and Maf expression. B: The relative expression of v-Jun and v-Maf in negative control, GFP control, and PTTG-overexpressed HEK293 cells by qRT-PCR. The relative expression values are mean ± SD (n = 3). The numbers above the bar represent the fold change of treatment with control group.

Figure 5

The Ingenuity pathway analysis of differentially expressed genes interlinked with the v-Jun regulation by PTTG.

Figure 6

Schematic diagram of PTTG's role in different pathways.

Figure 7

The Ingenuity pathway analysis of differentially expressed genes interlinked with the v-Maf regulation by PTTG.

Figure 8

PTTG up-regulates Histone gene expressions. A: Normalized expressions of H2be, H2bo, H2ac, and Hic in negative control, GFP control, and PTTG-overexpressed HEK293 cells by microarray analysis. Normalized expression values are mean ± SD (n = 3). The numbers above the bar represent the fold change of treatment with control group.

Figure 9

PTTG up-regulates Histone gene expressions. B: The relative expression of H2be, H2bo, H2ac, and Hic in negative control, GFP control, and PTTG-overexpressed HEK293 cells by qRT-PCR. The relative expression values are mean ± SD (n = 3). The numbers above the bar represent the fold change of treatment with control group.

Figure 10

The Ingenuity pathway analysis of differentially expressed genes interlinked with the Histone family gene regulation by PTTG.