Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis

Abstract

This study utilized both cDNA microarray and two-dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six-paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene-expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two-dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid-binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two-dimensional protein analysis via its cellular process-level approach. Therefore, a valuable prognostic candidate gene with relevance to disease-specific pathogenesis can be found at cellular process levels.

Figure 1

Hierarchical cluster analysis of uterine leiomyoma. (a) Type I assay: All the data were median centred and clustered using a hierarchical clustering. Levels of intensity of red squares correlate with the degree of gene expression; conversely, green squares compare the down-expression at a scale relative to the colour intensity. A cluster image representing 44 of the cDNAs is shown. (b) Type II assay (differential expression between uterine leiomyoma and normal myometrium): Type II analysis was used to identify the genes differentially expressed in the uterine leiomyoma by universal control cell lines, selecting 44 genes. Those genes showing statistically significant differences between the two groups are shown as a cluster image. The dendrogram indicates uterine leiomyoma in red and normal myometrium in green. The normal myometrims cluster together, as do the leiomyoma. The column on the left of the two-dimensional view indicates downregulated genes in myometrium in green and upregulated in red; conversely, the column on the right indicates downregulated genes in leiomyoma in green and upregulated in red.

Figure 2

Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of selected genes confirmed differential expression of cDNA microarray. Total RNA obtained from normal myometrium (lane 1) and leiomyoma (lane 2) was subjected to RT-PCR assays as described in Materials and methods.

Figure 3

Uterine leiomyoma protein products on two-dimensional gels. Identification of the proteins in 30 spots, which are indicated by red arrowheads (a, myometrium) and blue arrowheads (b, leiomyoma), is summarized in Table 2. The two-dimensional gel patterns indicated that out of the 30 spots.