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. 2020 Nov 7;12:168. doi: 10.1186/s13148-020-00961-y

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — STXBP5-AS1 epigenetically regulated neighboring gene ADGB transcription by binding to EZH2. a–c The expression level of ADGB in STXBP5-AS1 overexpressing and downregulated -PANC-1 and Mia PaCa-2 cells was determined by qRT-PCR and western blot. d, e Distribution of STXBP5-AS1 in PANC-1 and Mia PaCa-2 cells was determined by qRT-PCR. f RIP assay was performed to determine the association between STXBP5-AS1 and EZH2. The fold enrichment of STXBP5-AS1 in PANC-1 and Mia PaCa-2 cells with antibodies against EZH2 was relative to non-specific IgG control. g ChIP-PCR assay was performed to determine the interaction between ADGB promoter and EZH2 with antibodies against EZH2 or IgG in PANC-1 and Mia PaCa-2 cells. h, i ChIP-PCR assay was performed to determine EZH2 enrichment at ADGB promoter region with antibodies against EZH2 or IgG in PANC-1 and Mia PaCa-2 cells transfected with STXBP5-AS1 siRNAs (si-STXBP5-AS1-1 and si-STXBP5-AS1-2) or negative control siRNA (si-NC). (J and K) ChIP-PCR assay was performed to determine EZH2 enrichment at ADGB promoter region with antibodies against EZH2 or IgG in PANC-1 and Mia PaCa-2 cells transfected with STXBP5-AS1 plasmid (pSin-STXBP5-AS1) or empty vector (pSin-vector). (L) The bisulfite sequencing PCR analysis (BSP) of the methylation levels of ADGB in 5-Aza-CdR-treated or STXBP5-AS1-downregulated PANC-1 and Mia PaCa-2 cells. (M) The bisulfite sequencing PCR analysis (BSP) of the methylation levels of ADGB in STXBP5-AS1-upregulated, STXBP5-AS1-upregulated plus EZH2 knockdowned or STXBP5-AS1-upregulated plus 5-Aza-CdR-treated PANC-1 and Mia PaCa-2 cells. The data represent the mean ± SD from three independent experiments. *P < 0.05; ** P < 0.01, Student’s t test