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