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Fig. 5 | Molecular Cancer

Fig. 5

From: LncRNA SATB2-AS1 inhibits tumor metastasis and affects the tumor immune cell microenvironment in colorectal cancer by regulating SATB2

Fig. 5

SATB2-AS1 directly binds to WDR5 and GADD45A, and downregulation of SATB2 in CRC is due to high DNA methylation and loss of histone H3K4me3. a, Representative image of silver-stained PAGE gels showing separated proteins that were pulled down using biotin-labeled SATB2-AS1. A red arrow indicates WDR5, and a green arrow indicates GADD45A. b, Upper panel, western blot of the proteins from antisense SATB2-AS1 and SATB2-AS1 pull-down assays. Lower panel, western blot of WDR5 and GADD45A in samples pulled down by full-length or truncated SATB2-AS1 (Δ1: 1–500, Δ2: 501–1000, Δ3: 1001-1500, Δ4: 1501–2000, Δ5: 2001–2557). c, RNA immunoprecipitation with an anti-WDR5 antibody was used to assess endogenous WDR5 binding to RNA in CRC cells, IgG was used as the control. SATB2-AS1 and GAPDH levels were determined by qRT-PCR and are presented as fold enrichment in WDR5 relative to input. The RIP efficiency of WDR5 protein was detected by western blot. d, Left panel, DNA methylation analysis of SATB2 in CRC and corresponding normal tissues generated from the 450 k DNA methylation data in the TCGA database. Red in the heatmap denotes upregulation of methylation level; blue denotes downregulation. Each square represents a single CpG site, and each row represents a tissue sample. A scheme representing the SATB2 and SATB2-AS1 locus including the sites of each transcript, is displayed above. Right panel, statistical analysis of differences in methylation levels between tumors and adjacent normal tissues in the R3 and R4 regions. e, Bisulfite sequencing was performed to detect CpG island methylation statuses in R3 and R4 regions in ten paired CRC tissues and adjacent normal tissues. Representative images and corresponding statistical plots were presented. Black solid and white hollow circles represent methylated and unmethylated CpG sites, respectively. f, Left panel, analysis of H3K4me3 ChIP-seq data of colorectal tissues and CRC cells in the SATB2 locus. Right panel, the relative H3K4me3 levels of the SATB2 promoter in ten CRC tissues and matched adjacent colorectal tissues were evaluated by ChIP-qPCR. *, P < 0.05; **, P < 0.01 and ***, P < 0.001

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