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. Author manuscript; available in PMC: 2018 Dec 20.
Published in final edited form as: Oncogene. 2015 Feb 23;34(44):5548–5559. doi: 10.1038/onc.2015.10

Figure 5.

Figure 5.

Epigenetic alterations coinciding with CSC-mediated repression of miR-217. (*p<0.05; **p<0.01)

A: qRT-PCR analysis demonstrating that CSC does not decrease DA732292 and miR-216a expression levels in Het-1A and EACC.

B: Upper panel: Schematic depiction demonstrating CpG island approximately 5 kb up-stream of the miR-217 genomic locus. 25 NF1 binding sites are densely distributed within or adjacent to this CpG island. Lower panel: results of bisulfite sequencing experiments demonstrating increased DNA methylation within the miR-217 associated CpG island in EsC2 cells relative to Het-1A. Basal levels of DNA methylation in this CpG island are higher in EACC relative to IEEC. CSC increased DNA methylation in this CpG island in Het-1A as well as EACC.

C: MSP analysis demonstrating increased methylation within the miR-217 associated CpG island in EACC relative to IEEC.

D: Quantitative ChIP analysis demonstrating that CSC decreases H3K4me3 and increases H3K27me3 levels within the miR-217 associated CpG island. These findings are consistent with results of bisulfite sequencing and MSP experiments shown above.

E: qRT-PCR analysis demonstrating that DAC (100 nM x 72h) significantly increases expression of miR-217 in EACC but not Het-1A cells. DZNep (5 uM) had minimal effects when administered alone, but augmented DAC-mediated induction of miR-217 in OE19 cells.

F: MeDIP analysis of DNA methylation profiles within the CpG island upstream of miR-217 in Het-1A, EsC1, and EsC2 cells treated with CSC and/or DAC (100 nM x72h). CSC dramatically increased methylation in this CpG island. DAC significantly abrogated CSC-mediated CpG methylation in these cells. CSC and DAC had no effect on DNA methylation levels within the UBE2B promoter (negative methylation control) or the H19 ICR (positive methylation control).

G: Representative MSP analysis of primary EAC and paired adjacent normal esophageal tissues demonstrating methylation of the miR-217 associated CpG island in EAC but not normal esophageal mucosa. Methylated PCR products were more prominent in EAC from smokers/former smokers relative to non-smokers, suggesting increased methylation of the miR-217 CpG island in patients with a history of cigarette abuse.