Overexpression of Ribosomal RNA in the Development of Human Cervical Cancer Is Associated with rDNA Promoter Hypomethylation.

Hong Zhou,Juan Zhang,Hao Zhang,Lijia Li,Haoli Hou,Qiongying Lv,Yapei Wang,Fei Gao,Qing Wang,Wei Zhang,Xuefeng Liu

doi:10.1371/journal.pone.0163340

Abstract

The ribosomal RNA (rRNA) gene encodes rRNA for protein synthesis. Aberrant expression of the rRNA gene has been generally observed in tumor cells and levels of its promoter methylation as an epigenetic regulator affect rRNA gene transcription. The possible relationship between expression and promoter methylation of rDNA has not been examined in human clinical cervical cancer. Here we investigate rRNA gene expression by quantitative real time PCR, and promoter methylation levels by HpaII/MspI digestion and sodium bisulfite sequencing in the development of human cervical cancer. We find that indeed rRNA levels are elevated in most of cervical intraepithelial neoplasia (CIN) specimens as compared with non-cancer tissues. The rDNA promoter region in cervical intraepithelial neoplasia (CIN) tissues reveals significant hypomethylation at cytosines in the context of CpG dinucleotides, accompanied with rDNA chromatin decondensation. Furthermore treatment of HeLa cells with the methylation inhibitor drug 5-aza-2’-deoxycytidine (DAC) demonstrates the negative correlation between the expression of 45S rDNA and the methylation level in the rDNA promoter region. These data suggest that a decrease in rDNA promoter methylation levels can result in an increase of rRNA synthesis in the development of human cervical cancer.

Highlights

In eukaryotes, 45S ribosomal RNA gene is arranged in arrays of head-to-tail tandem repeats known as nucleolar organizer regions (NORs) [1,2,3,4]
Our data showed that the 45S rDNA transcription was increased in the majority of clinical cervical intraepithelial neoplasia (CIN) specimens and HeLa cell lines treated with the methylation inhibitor 5-aza-2’-deoxycytidine (DAC) whereas the rDNA promoter region revealed significant hypomethylation, accompanied with rDNA chromatin decondensation. These results suggested that ribosomal RNA (rRNA) synthesis and rDNA promoter methylation at CpG islands were inversely correlated in the development of human cervical cancer
To determine whether the 45S rDNA transcript was elevated in development of human cervical cancer, total RNA was extracted from a series of non-cancerous and cervical precancerous fresh frozen tissue samples which were obtained from ten cervical epithelial cell abnormality patients, and results from real time quantitative reverse transcriptase PCR for the 45S precursor (Fig 2A and 2B) showed that pre-rRNA levels were significantly higher in CIN samples as compared with non-cancerous samples

Summary

Introduction

45S ribosomal RNA (rRNA) gene (rDNA) is arranged in arrays of head-to-tail tandem repeats known as nucleolar organizer regions (NORs) [1,2,3,4]. The number of rRNA gene copies varies greatly among organisms from fewer than 100 to more than 10 000. The rDNA clusters in mammals include the intergenic spacer and a pre-rRNA coding region. Approximately 400 copies of rRNA genes are distributed along five pairs of acrocentric chromosomes 13, 14, 15, 21 and 22, but only parts of genes are active [5]. The rRNA genes are transcribed by RNA polymerase I (Pol I) to produce a long rRNA precursor that is . October 3, 2016 element; Pol I, RNA polymerase I; PCR, polymerase chain reaction PLOS ONE | DOI:10.1371/journal.pone.0163340 October 3, 2016 element; Pol I, RNA polymerase I; PCR, polymerase chain reaction

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Conclusion