Fig. 6

LINC01977-dependent SMAD3/CBP/P300 complex epigenetically up-regulates ZEB1, the central switch of EMT process, in LUAD cells. A The circos plots showing dysregulated RNAs found in RNA-seq analysis in A549 cells, up-regulated (pcDNA3.1 vs LINC01977) and down-regulated (LINC01977-ASO vs Scramble). B, C Significantly dysregulated genes were validated by qRT-PCR in A549 cells. D Kaplan–Meier survival analysis of ZEB1 expression in LUAD patients from JACOB-00812-HLM dataset. E Correlation analysis of SMAD3 and LINC01977 in TCGA-LUAD dataset. F SMAD3-binding motif analysis was performed on ZEB1 promoter via JASPAR database. (G) Schematic representation of SMAD3-binding sites on ZEB1 promoter and primers design strategy. H Transcription activity of ZEB1 was elevated in A549 cells treated with TGF-β (10 ng/mL), and significantly promoted by transfected with LINC01977 and SMAD3 followed by treatment with TGF-β (10 ng/mL) as indicated. (I) Mutant-binding site #4 (MT4) diminished the transcriptional activity of LINC01977 in A549 cells. Transcriptional activity of LINC01977 was elevated by treated with TGF-β (10 ng/mL) and promoted by overexpression of LINC01977 or SMAD3, also decreased by absent of LINC01977 or SMAD3. J ChIP assays suggested that only #4 sub-region of ZEB1 promoter was co-occupied by CBP, P300, SMAD3 and H3K27ac. (K) ChIP assays suggested that co-occupancy of #4 sub-region of ZEB1 promoter by CBP, P300, SMAD3, and H3K27ac was promoted by LINC01977 overexpression. Data in A-C and H–K are representative of three independent experiments and presented as mean ± S.D., n = 3 biologically independent samples, and the P value was determined by a two-tailed unpaired Student’s t test and one-way ANOVA, respectively. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s. not significantly