Effect Of DNA Methylation Inhibitors 5-Azacytidine And 5-Aza-2'-deoxycytidine On The Expression Of Coding And Non-coding RNAs

Backgroud DNA methylation occurs almost exclusively on a cytosine in a CpG dinucleotide, and is achieved by the addition of a methyl group to the 5 position of a cytosine ring mediated by DNA methyl transferases (DNMTs). The CpG sites are asymmetrically distributed into CpG poor regions and dense regions called "CpG islands", which are often located in the promoter regions of approximately half of all protein-coding genes. CpG islands normally remain unmethylated, while the most CpG poor regions are normally methylated. DNA methylation dysregulation in cancer includes abnormal global DNA hypomethylation particularly in CpG poor region and repeat regions, which induces oncogene activation and chromosomal instability, and promoter hypermethylation of CpG islands, which leads to the silencing of some tumor suppressor genes.5-Azacytidine (5-Aza-CR) and 5-Aza-2'-deoxycytidine (5-Aza-CdR) are two well-known DNA methylation inhibitors, and they have been approved by the Food and Drug Administration for the treatment of myelodysplastic syndrome. Both drugs are thought to exert their effects after incorporation into DNA and RNA by covalent binding of DNA methyltransferase (DNMT). While 5-aza-CdR is only incorporated into only DNA,5-aza-CR is incorporated into both DNA and RNA.5-aza-CR is the first drug to show a survival benefit in patients with myelodysplastic syndrome. Surprisingly, the deoxyribonucleoside analog 5-aza-CdR did not have a similar positive effect on survival in a large clinical trial. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacity of these drugs to regulate the expression of coding and non-coding RNA, and could be one possible explanation for the differences in treatment outcome.Objective To investigate whether the different metabolism mechanisms of 5-Aza-CR and 5-Aza-CdR influence the capacity of these drugs to regulate the expression of coding and non-coding RNA. Methods A hematopoietic (P39; MDS/AML) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-aza-CR and 5-aza-CdR. The medium was changed after 24 hours, and cells were collected 1 day (Day 2) and 7 days (Day 8) after the drug had been removed. High-throughput screening approaches, including mRNA array, miRNA array and ncRNA array, were utilized to examine the expression of mRNA, miRNA and longer non-coding RNA (ncRNA). Differential expression analysis was done using Limma. Real-time RT-PCR was used to detect the expression of XAGE1D. DNA methylation status of XAGE1D was measured by Methylation-Specific Single Nucleotide Primer Extension (Ms-SNuPE).Results Both drugs effect the expression of all RNA species:mRNA, microRNA and long non-coding RNA. In general,5-aza-CdR treatment upregulated considerably more RNAs than 5-aza-CR, particulaly on day 8, and more RNAs were downregulated on day 2 by 5-aza-CR. A large proportion of RNAs upregulated on day 8 after 5-aza-CR treatment were also upregulated by 5-aza-CdR. Among those upregulated genes, many are belonged to the group of cancer testis antigens (CTAs). The upregulation of CTAs coincided with upregulation of CTCFL (BORIS), which may act as a transcriptional regulator of CTAs. On day 8, multiple genes from the interleukin-and interferon pathways were upregulated by both drugs, indicating a role of induction of an inflammatory and/or immunological response. A more detailed analysis immediately after treatment revealed diverse effects of each drug:On day 2, 5-aza-CR led to a remarkable down regulation of small non-coding RNAs, and significant upregulation of tRNA synthetases and certain genes involved in amino acid metabolism was observed in T24 cells.Conclusion Significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.Objective To explore whether miR-886 transcript is vault RNA 2. And to study whether its expression can be regulated by 5-Aza-CR and 5-Aza-CdR.Methods Rapid amplification of cDNA ends (RACE) was used to find the transcription start site and 3'end of mir-886 transcript. The expression of mature miR-886 was detected by stem-loop RT-PCR in P39, LD419, UROtsa, T24, and UMUC3. DNA methylation status of potential promoter of miR-886 transcript (vault RNA2) was measured by Methylation-Specific Single Nucleotide Primer Extension (Ms-SNuPE). T24 cells were transfected twice with Drosha siRNAs, and collected at 72hr after the second transfection. Expression of Drosha mRNA was detected by real-time RT-PCR, and western blot was used for protein expression. Stem-loop RT-PCR was used to measure the expression of mature miR-886 and miR-21. T24 cells were treated with a-amanitin, and harvested at 24 hr and 48hr after treatment. The expression of miR-886 transcript was detected by RT-PCR. Real-time RT-PCR and Northern blot were used to measure the expression of miR-886 transcript.Results Although miR-886 transcript is shorter than predicted pri-miRNA and the transcription product has the appropriate size to form a pre-miRNA hairpin, miR-886 transcript is transcribed by RNA polymerase III and not by RNA polymerase II and mature mir-886 production is independent of Drosha. These results showed miR-886 transcipt is neither pri-miRNA nor pre-miRNA. Futhermore, Northern blot results indicated that instead of miR-886 vault RNA 2 is the major products which can produce miR-886 or miRNA-like small RNA. In general, Vault RNA 2 is expressed in normal cell lines such as LD419, UROtsa. It was silenced by its promoter methylation in UMUC3 and P39 cancer cell lines. The expression of vault RNA2 can be restored by 5-aza-CR and 5-aza-CdR treatment accompanied by reduction of methylation in P39 and UMUC3 cancer cell lines,Conclusion miR-886 transcript is vault RNA 2, which can produce small RNA. miR-886-3p and miR-886-5p. Our study has revealed for the first time that DNA methylation can silence vault RNA2 expression. DNA methylation inhibitors. 5-aza-CR and 5-aza-CdR, can restore the expression of methylation silenced vault RNA 2 in cancer cell lines. Although vault RNA 2 is commonly silenced in cancer cell lines, the role of vault RAN2 during the tumirgenesis is still under investigation.