Journal: Cell Death & Disease
Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2
doi: 10.1038/s41419-025-08380-8
Figure Lengend Snippet: a SEs were calculated and ranked by H3K27ac signal using ROSE algorithm. And a proximal SE within LINC00973 gene locus were identified in 3 HNSCC cell lines (Cal27, HN12, and HSC3). b Genomic tracks plot displayed the H3K27ac enrichment (including 6 cell lines, 2 primary HNSCC and 1 human normal oral mucosa (NOM) tissues), BRD4, and EP300 binding and chromatin accessibility at the LINC00973-SE region. Four individual enhancer fragments within LINC00973-SE were observed and referred as E1-E4. c The H3K27ac, BRD4 and EP300 enrichment on E1-E4 or E-NC were measured by ChIP-qPCR in Cal27 and HN6 cells. d , e The expression changes of LINC00973 were measured by qRT-PCR and the binding changes of H3K27ac, BRD4, and EP300 on E1-E4 were determined by ChIP-qPCR in Cal27 and HN6 cells treated with JQ1 (1 μM, 12 h) or NEO2734 (1 μM, 12 h). f Representative FISH staining of LINC00973 and IHC staining of EN2 in PDX samples treated with vehicle, JQ1 or NEO2734. g Schematic diagram showing the procedure of E1-E4 repression by dCas9-KRAB-MeCP2 system. h LINC00973 expression in Cal27-dCas9 and HN6-dCas9 cells with repressed LINC00973 enhancer activity were assessed by qRT-PCR. Cell proliferation, migration and invasion were significantly suppressed following sgE2 treatment as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l De novo motif discovery at LINC00973-SE regions via HOMER algorithm. m LINC00973 expression was detected by qRT-PCR in Cal27 and HN6 cell with/without FOSL1 silencing. n , o co-IP assays revealed the endogenous FOSL1 protein interacted with Rpb1 (RNA pol II subunit) and BRD4 in both Cal27 and HN6 cells ( i ), while FOSL1-depletion disrupted these interactions ( j ). p The FOSL1 binding enrichments at E2 region in SCC1 with/without FOSL1 knockdown were displayed by track plot. q FOSL1 binding on E1-E4 or E-NC regions in Cal27 and HN6 transfected with si-FOSL1 were measured by ChIP-qPCR. r The core DNA sequences of E2 or E-NC were subcloned into pGL3 vectors and the luciferase activities were detected in Cal27 and HN6 cells transfected with si-FOSL1 or control siRNAs. s Schematic model of LINC00973 transcription regulation: cis -activation by LINC00973-SE and trans -regulation by AP-1/FOSL1. Data were presented as mean ± SD, # P ≥ 0.05,* P < 0.05, ** P < 0.01, Student’s t test.
Article Snippet: Epigenetic inhibitors JQ1 (HY-13030) and NEO2734 (HY-136938) were purchased from MedChemExpress (USA), dissolved in DMSO, and administered at indicated concentrations.
Techniques: Binding Assay, ChIP-qPCR, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry, Activity Assay, Migration, CCK-8 Assay, Co-Immunoprecipitation Assay, Knockdown, Transfection, Luciferase, Control, Activation Assay
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