Fig. 5

LINC02273 transcriptionally activates AGR2 expression. a Flow chart of LINC02273 targets selection. Differential genes were selected by comparing WT to LINC02273 KO MDA-MB-231 cells. LINC02273 DNA binding sites were selected by merging odd and even pool of ChIRP-seq. RNA-DNA binding in − 2000-200 bp to transcriptional start site (TSS) were selected. b Venn diagram of LINC02273 target selection. c KEGG analysis of the 182 candidate genes. d LINC02273 DNA binding were confirmed by ChIRP-qPCR with indicated probes. LacZ probes were used as negative control. e RT-qPCR analysis of candidate genes in WT and LINC02273 KO cells. log2 fold change were shown. n = 3, biological replicates. f RT-qPCR analysis of AGR2 expression in WT and LINC02273 KO cells. n = 3, biological replicates. g Immunoblotting of AGR2 after LINC02273 KO. h Luciferase reporter assay with LINC02273 overexpression. Left, schematic view of LINC02273 binding site on AGR2 TSS. Truncations were ligated to pGL3-basic vector. Right, luciferase assay was performed with co-transfection of pcDNA3.1-LINC02273 and full length or truncated reporter vectors. i Representative images of ChIRP-seq and ChIP-seq results with indicated probes or antibodies on AGR2 genomic region. The peak range of each track were indicated in the square brackets. AGR2-peak represents the DNA region that cloned and used in (h). j Transwell migration and invasion assay with ectopically overexpressed AGR2. Scale bars: 100 μm. n = 3, biological replicates. k Representative images of BLI, 8 weeks after tail vein injection of indicated cells. Photon flux was calculated and compared (n = 6, Mann-Whitney test). l H&E staining of lung metastasis. 40× images of the red rectangular regions were shown. Scale bars: black, 2 mm; pink, 100 μm. For (d-f), (h), and (j-k), means ± s.e.m. are shown, and P values were determined by independent sample t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant