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Discovery pipeline for epigenetically deregulated miRNAs in cancer: integration of primary miRNA transcription.

Hulf T, Sibbritt T, Wiklund ED, Bert S, Strbenac D, Statham AL, Robinson MD, Clark SJ - BMC Genomics (2011)

Bottom Line: Cancer is commonly associated with widespread disruption of DNA methylation, chromatin modification and miRNA expression.We find miR-205, miR-21, and miR-196b to be epigenetically repressed, and miR-615 epigenetically activated in prostate cancer cells.We show that detecting changes in primary miRNA transcription levels is a valuable method for detection of local epigenetic modifications that are associated with changes in mature miRNA expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Epigenetics Laboratory, Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia.

ABSTRACT

Background: Cancer is commonly associated with widespread disruption of DNA methylation, chromatin modification and miRNA expression. In this study, we established a robust discovery pipeline to identify epigenetically deregulated miRNAs in cancer.

Results: Using an integrative approach that combines primary transcription, genome-wide DNA methylation and H3K9Ac marks with microRNA (miRNA) expression, we identified miRNA genes that were epigenetically modified in cancer. We find miR-205, miR-21, and miR-196b to be epigenetically repressed, and miR-615 epigenetically activated in prostate cancer cells.

Conclusions: We show that detecting changes in primary miRNA transcription levels is a valuable method for detection of local epigenetic modifications that are associated with changes in mature miRNA expression.

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Related in: MedlinePlus

Vailidation of pri-miRNA expression. Quantitative TaqMan® RT-PCR of pri-miRNA expression in LNCaP and PrEC cells +/- 5-Aza-CdR treatment (n = 6, +/-SD, *p <0.05). Pri-miRNA RT-PCR assays followed established TaqMan® workflows (Applied Biosystems). Briefly, total RNA was used for first-strand cDNA synthesis using 1 μg RNA Superscript reverse transcriptase (Invitrogen). cDNA was then subjected to 40 cycles of amplification using an ABI7900 instrument (Applied Biosystems). Data was normalized to 18S levels (2-dCt).
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Figure 3: Vailidation of pri-miRNA expression. Quantitative TaqMan® RT-PCR of pri-miRNA expression in LNCaP and PrEC cells +/- 5-Aza-CdR treatment (n = 6, +/-SD, *p <0.05). Pri-miRNA RT-PCR assays followed established TaqMan® workflows (Applied Biosystems). Briefly, total RNA was used for first-strand cDNA synthesis using 1 μg RNA Superscript reverse transcriptase (Invitrogen). cDNA was then subjected to 40 cycles of amplification using an ABI7900 instrument (Applied Biosystems). Data was normalized to 18S levels (2-dCt).

Mentions: To validate the changes in primary miRNA expression, we performed pri-miRNA TaqMan® qPCR on the four miRNAs that met all our criteria. These assays confirmed the repression of pri-miR-205 and pri-miR-21, and over expression of pri-miR-615 in LNCaP cells (Figure 3A,B,D). There was no significant change in pri-miR-196b between LNCaP and PrEC, however treatment with 5-Aza-CdR led to overexpression in PrEC cells (Figure 3C). 5-Aza-CdR led to overexpression of pri-miR-21 and pri-miR-615 in LNCaP, as would be predicted by their DNA methylation status (Figure 3B,D). However, reactivation of pri-miR-205 by 5-Aza-CdR was not significant (Figure 3A).


Discovery pipeline for epigenetically deregulated miRNAs in cancer: integration of primary miRNA transcription.

Hulf T, Sibbritt T, Wiklund ED, Bert S, Strbenac D, Statham AL, Robinson MD, Clark SJ - BMC Genomics (2011)

Vailidation of pri-miRNA expression. Quantitative TaqMan® RT-PCR of pri-miRNA expression in LNCaP and PrEC cells +/- 5-Aza-CdR treatment (n = 6, +/-SD, *p <0.05). Pri-miRNA RT-PCR assays followed established TaqMan® workflows (Applied Biosystems). Briefly, total RNA was used for first-strand cDNA synthesis using 1 μg RNA Superscript reverse transcriptase (Invitrogen). cDNA was then subjected to 40 cycles of amplification using an ABI7900 instrument (Applied Biosystems). Data was normalized to 18S levels (2-dCt).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037319&req=5

Figure 3: Vailidation of pri-miRNA expression. Quantitative TaqMan® RT-PCR of pri-miRNA expression in LNCaP and PrEC cells +/- 5-Aza-CdR treatment (n = 6, +/-SD, *p <0.05). Pri-miRNA RT-PCR assays followed established TaqMan® workflows (Applied Biosystems). Briefly, total RNA was used for first-strand cDNA synthesis using 1 μg RNA Superscript reverse transcriptase (Invitrogen). cDNA was then subjected to 40 cycles of amplification using an ABI7900 instrument (Applied Biosystems). Data was normalized to 18S levels (2-dCt).
Mentions: To validate the changes in primary miRNA expression, we performed pri-miRNA TaqMan® qPCR on the four miRNAs that met all our criteria. These assays confirmed the repression of pri-miR-205 and pri-miR-21, and over expression of pri-miR-615 in LNCaP cells (Figure 3A,B,D). There was no significant change in pri-miR-196b between LNCaP and PrEC, however treatment with 5-Aza-CdR led to overexpression in PrEC cells (Figure 3C). 5-Aza-CdR led to overexpression of pri-miR-21 and pri-miR-615 in LNCaP, as would be predicted by their DNA methylation status (Figure 3B,D). However, reactivation of pri-miR-205 by 5-Aza-CdR was not significant (Figure 3A).

Bottom Line: Cancer is commonly associated with widespread disruption of DNA methylation, chromatin modification and miRNA expression.We find miR-205, miR-21, and miR-196b to be epigenetically repressed, and miR-615 epigenetically activated in prostate cancer cells.We show that detecting changes in primary miRNA transcription levels is a valuable method for detection of local epigenetic modifications that are associated with changes in mature miRNA expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Epigenetics Laboratory, Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia.

ABSTRACT

Background: Cancer is commonly associated with widespread disruption of DNA methylation, chromatin modification and miRNA expression. In this study, we established a robust discovery pipeline to identify epigenetically deregulated miRNAs in cancer.

Results: Using an integrative approach that combines primary transcription, genome-wide DNA methylation and H3K9Ac marks with microRNA (miRNA) expression, we identified miRNA genes that were epigenetically modified in cancer. We find miR-205, miR-21, and miR-196b to be epigenetically repressed, and miR-615 epigenetically activated in prostate cancer cells.

Conclusions: We show that detecting changes in primary miRNA transcription levels is a valuable method for detection of local epigenetic modifications that are associated with changes in mature miRNA expression.

Show MeSH
Related in: MedlinePlus