Ribosomal RNA (rRNA) expression, one of the most important factors regulating ribosome production, is primarily controlled by a CG-rich 45 S
rDNA promoter. However, the DNA methylation state of the 45 S
rDNA promoter, as well as its effect on rRNA gene expression in types of human
cancers is controversial. In the present study we analyzed the methylation status of the
rDNA promoter (-380 to +53 bp) as well as associated rRNA expression levels in
breast cancer cell lines and
breast tumor-normal tissue pairs. We found that the aforementioned regulatory region was extensively methylated (74-96%) in all cell lines and in 68% (13/19
tumor-normal pairs) of the
tumors. Expression levels of rRNA transcripts 18 S, 28 S, 5.8 S and 45 S external transcribed spacer (45 S ETS) greatly varied in the
breast cancer cell lines regardless of their methylation status. Analyses of rRNA transcript expression levels in the
breast tumor and normal matched tissues showed no significant difference when normalized with
TBP. On the other hand, using the geometric mean of the rRNA expression values (GM-rRNA) as reference enabled us to identify significant changes in the relative expression of rRNAs in the tissue samples. We propose GM-rRNA normalization as a novel strategy to analyze expression differences between rRNA transcripts. Accordingly, the
18S rRNA/GM-rRNA ratio was significantly higher whereas the
5.8S rRNA/GM-rRNA ratio was significantly lower in
breast tumor samples than this ratio in the matched normal samples. Moreover, the
18S rRNA/GM-rRNA ratio was negatively correlated with the 45 S
rDNA promoter methylation level in the normal breast tissue samples, yet not in the
breast tumors. Significant correlations observed between the expression levels of rRNA transcripts in the normal samples were lost in the
tumor samples. We showed that the expression of rRNA transcripts may not be based solely on promoter methylation.
Carcinogenesis may cause dysregulation of the correlation between spliced rRNA expression levels, possibly due to changes in rRNA processing, which requires further investigation.