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Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.

Ding XL, Yang X, Liang G, Wang K - Sci Rep (2016)

Bottom Line: By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression.We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment.Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17.

View Article: PubMed Central - PubMed

Affiliation: Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China.

ABSTRACT
DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2'-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials.

No MeSH data available.


Related in: MedlinePlus

Dynamic transcriptome changes induced by 5-Aza-CdR treatment.(a) The number of differentially expressed genes across different time points. (b) Venn diagram showing overlapped differentially expressed genes found in each time point. (c) The number of up and down regulated genes found in the two RNA types: protein coding RNAs and lncRNAs. (d) Tumor suppressor gene expression pattern. (Y axis indicates normalized expression, X axis indicates different 5-Aza-CdR exposure time, A and B indicated two replicates) (e) Hierarchical clustering of differentially expressed genes.
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f1: Dynamic transcriptome changes induced by 5-Aza-CdR treatment.(a) The number of differentially expressed genes across different time points. (b) Venn diagram showing overlapped differentially expressed genes found in each time point. (c) The number of up and down regulated genes found in the two RNA types: protein coding RNAs and lncRNAs. (d) Tumor suppressor gene expression pattern. (Y axis indicates normalized expression, X axis indicates different 5-Aza-CdR exposure time, A and B indicated two replicates) (e) Hierarchical clustering of differentially expressed genes.

Mentions: RNA-seq data generated from untreated UM-UC-3 cells (control) and cells collected from four time points (Day 5, Day 9, Day 13 and Day 17) were aligned to human genome using Tophat218 with GENCODE annotation (GRCh37.p13, GENCODE release 19). For all samples, we obtained more than 92% mapping ratio, which indicated high quality and reliability of the sequencing data. Differentially expressed (DE) genes were identified by comparing RNA-seq data obtained from each treatment with untreated cells. In total, 1315, 1344, 1393 and 1612 DE genes were found on Day 5, Day 9, Day 13 and Day 17, respectively (Fig. 1a). Among those DE genes, 847 of them were shared in all four time points (Fig. 1b). About 85% (Day 5: 90%, Day 9: 85%, Day 13: 84%, Day 17: 85%) DE genes were up regulated after 5-Aza-CdR treatment. Furthermore, the numbers of up and down regulated DE genes were positively correlated with the treatment time of 5-Aza-CdR and the most abundant DE genes were always found after 17 days treatment (Fig. 1a).


Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.

Ding XL, Yang X, Liang G, Wang K - Sci Rep (2016)

Dynamic transcriptome changes induced by 5-Aza-CdR treatment.(a) The number of differentially expressed genes across different time points. (b) Venn diagram showing overlapped differentially expressed genes found in each time point. (c) The number of up and down regulated genes found in the two RNA types: protein coding RNAs and lncRNAs. (d) Tumor suppressor gene expression pattern. (Y axis indicates normalized expression, X axis indicates different 5-Aza-CdR exposure time, A and B indicated two replicates) (e) Hierarchical clustering of differentially expressed genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835787&req=5

f1: Dynamic transcriptome changes induced by 5-Aza-CdR treatment.(a) The number of differentially expressed genes across different time points. (b) Venn diagram showing overlapped differentially expressed genes found in each time point. (c) The number of up and down regulated genes found in the two RNA types: protein coding RNAs and lncRNAs. (d) Tumor suppressor gene expression pattern. (Y axis indicates normalized expression, X axis indicates different 5-Aza-CdR exposure time, A and B indicated two replicates) (e) Hierarchical clustering of differentially expressed genes.
Mentions: RNA-seq data generated from untreated UM-UC-3 cells (control) and cells collected from four time points (Day 5, Day 9, Day 13 and Day 17) were aligned to human genome using Tophat218 with GENCODE annotation (GRCh37.p13, GENCODE release 19). For all samples, we obtained more than 92% mapping ratio, which indicated high quality and reliability of the sequencing data. Differentially expressed (DE) genes were identified by comparing RNA-seq data obtained from each treatment with untreated cells. In total, 1315, 1344, 1393 and 1612 DE genes were found on Day 5, Day 9, Day 13 and Day 17, respectively (Fig. 1a). Among those DE genes, 847 of them were shared in all four time points (Fig. 1b). About 85% (Day 5: 90%, Day 9: 85%, Day 13: 84%, Day 17: 85%) DE genes were up regulated after 5-Aza-CdR treatment. Furthermore, the numbers of up and down regulated DE genes were positively correlated with the treatment time of 5-Aza-CdR and the most abundant DE genes were always found after 17 days treatment (Fig. 1a).

Bottom Line: By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression.We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment.Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17.

View Article: PubMed Central - PubMed

Affiliation: Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China.

ABSTRACT
DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2'-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials.

No MeSH data available.


Related in: MedlinePlus