Fig 2: Potential therapeutic role of LINC02273 in breast cancer. a Schematic view of ASO targets on LINC02273 mRNA. b Relative expression of LINC02273 48 h after ASO transfection were determined by RT-qPCR (normalized to GAPDH). n = 3, biological replicates. c Immunoblotting analysis of AGR2 expression after ASO transfection in MDA-MB-231 cells. GAPDH were served as loading control. n = 3 independent experiments. d RT-qPCR showed LINC02273 expression after ASO delivery without transfect reagents. ASO were added into LM2 wild type cells at the concentration as indicated. After 48 h, RNA was extracted, and RT-qPCR was performed. Normalized to GAPDH, n = 3, biological replicates. e Schematic view of ASO treatment xenograft model. LM2 WT cells were injected into mammary fat pad. Mice were randomized into 3 groups 14 days later. ASO were diluted in PBS and delivered through tail vein twice a week with the concentration of 10 nmol foreach mouse. ASO NC targeted no known sequence with the same length of ASO-1 and ASO-2 were used as negative control. Tumor sizes were measured during treatment. Mice were sacrificed on the day 45. BLI was performed ex vivo. f Tumor volumes were measured in the NC and ASO groups in the xenograft mouse model (n = 8, independent t-test). g Representative images of BLI of lungs and statistical analysis of photon flux. Scale bar: 4 mm. Statistical analysis of total photon flux is on the right (n = 8, Mann-Whitney test). h LINC02273 and AGR2 expression in xenograft tumor were determined by RT-qPCR (n = 8, Mann-Whitney test). i Schematic diagram of hnRNPL-LINC02273-AGR2 axis regulating breast cancer metastasis. For (b), (d), and (f-g), means ± s.e.m. are shown. For (b) and (d), P values were determined by independent sample t-tests. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: 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