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: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

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

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 Schematic diagram outlining the experimental design to identify and characterize downstream signaling pathways modulated by EN2 in HNSCC. b Differential analyses of KEGG pathways were performed in Cal27 and HN6 cells with/without EN2 silencing and the results were represented as scatter plot (left panel) and heatmap (right panel). KEGG pathway scores were calculated by GSVA method. c Correlation analyses between LINC00973 expression and KEGG_NOTCH_SIGNALING_PATHWAY scores (GSVA method) in TCGA-HNSC, GSE186775 and GSE65858 datasets. d The transcription activity of NOTCH1, the core member of NOTCH pathway, was measured by NOTCH1 luciferase reporter assays in Cal27 and HN6 with EN2 manipulations. e The protein abundance of NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with EN2 knockdown. f The endogenous NOTCH1, N1ICD and HEY1 protein expression was detected in Cal27 and HN6 cells followed by LINC00973 siRNAs or HN30 cells with LINC00973 overexpression, respectively. g NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/withoutEN2 overexpression. Cell proliferation, migration and invasion were significantly increased following LINC00973 overexpression but reduced by DAPT treatment as gauged by CCK-8 ( h ), wound healing ( i ) and Transwell invasion assays ( j ).Scale bar: 50 μm. k Representative H&E, IHC staining of FOSL1, EN2, and NOTCH1 as well as FISH staining of LINC00973, in serial sections of the same HNSCC clinical samples were shown. Scale bar: 50 μm. l The correlation among FOSL1, EN2 and NOTCH1 protein expression (IHC quantifications data) and LINC00973 RNA abundance (FISH signal intensity) were analyzed by using Spearman’s correlation. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

Techniques: Protein-Protein interactions, Expressing, Activity Assay, Luciferase, Quantitative Proteomics, Western Blot, Knockdown, Over Expression, Migration, CCK-8 Assay, Immunohistochemistry, Staining

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: The proximal SE in LINC00973 locus cis -activates LINC00973 transcription via recruiting AP-1/FOSL1, BRD4 and EP300; the cytoplasmically localized LINC00973 sponges miR-6756-3p to increase the mRNA half-life of EN2; EN2 activates downstream NOTCH signaling to modulate the aggressiveness of HNSCC.

Article Snippet: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

Techniques:

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 The relative expression of LINC00973 in normal human oral keratinocytes (HOK) cell and HNSCC cell lines were measured by qRT-PCR. b Subcellular RNA fraction and followed qRT-PCR assays were conducted to determine the localization of LINC00973 within Cal27 and HN6 cells. GAPDH and U6 served as the internal control of cytoplasm and nuclear, respectively. c RNA FISH assays showed LINC00973 mainly localized in cytoplasm in Cal27 and HN6 cells. Scale bar: 10 μm. d The knockdown efficiency of LINC00973 was determined by qRT-PCR in Cal27 and HN6 cells transfected si-LINC00973-1 (si-1) and si-LINC00973-2 (si-2). Cell proliferation, migration, and invasion was evaluated by CCK-8 ( e ), wound-healing ( f ), and Transwell invasion assays ( g ) in LINC00973-sliencing Cal27 and HN6 cells. Scale bar: 50 μm. h The protein abundance of indicated migration and invasion related markers was detected in LINC00973-deficient cells. i Schematic description of experimental procedures for the orthotopic floor-of-mouth xenograft model. j Representative image of the tumor masses with stable LINC00973 knockdown/control HN6 cells and the final volume of samples were recorded. Scale bar: 1 cm. k Representative IHC staining of Ki67 in xenograft tumors (left panel) and their quantification data were shown (right panel). Scale bar: 50 μm. l Representative H&E staining and IHC staining of CK5/6 in lymph nodes (left panel) from orthotopic tumor-bearing mice. The percentage of lymph nodes metastasis were shown and compared (right panel). Fisher’s exact test. Data shown here are mean ± SD from three independent experiments, ** P < 0.01, Student’s t or one-way ANOVA test.

Article Snippet: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Transfection, Migration, CCK-8 Assay, Quantitative Proteomics, Immunohistochemistry, Staining

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: Heatmap ( a ) and volcano plot ( b ) displayed the expression profile of DEGs following LINC00973 knockdown or control cells. The mRNA ( c ) and protein ( d ) expression of EN2 were measured in Cal27 and HN6 cells with/without EN2 knockdown. e , f The correlations between EN2 and LINC00973 RNA expression were assessed in the public HNSCC datasets ( GSE41613 and GSE42743 ) and our in-house samples. Spearman’s correlation. The mRNA ( g ) and protein ( h ) abundance of EN2 were measured in Cal27 and HN6 cells under four conditions. Cell proliferation, migration and invasion were significantly reduced following LINC00973 knockdown but restored by ectopic EN2 overexpression as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l The migration/invasion-related markers were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/without EN2 overexpression. m 1 × 10 5 HN6 cells with LINC00973 or/and EN2 manipulations were injected submucosally into the floor of the mouth. Fourteen days post-injection, tumor masses and cervical lymph nodes were harvested. Representative tumor masses images and estimated tumor volumes were showed. Scale bar: 1 cm. Representative IHC staining of Ki67 and EN2 in xenograft tumors ( n ) and their quantification data were shown ( o ). Scale bar: 50 μm. p Representative IHC staining of CK5/6 in lymph nodes (left panel) from orthotopic tumor-bearing mice among four indicated groups were displayed. The percentage of lymph nodes metastasis were shown and compared (right panel). Fisher’s exact test. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

Techniques: Expressing, Knockdown, Control, RNA Expression, Migration, Over Expression, CCK-8 Assay, Western Blot, Injection, Immunohistochemistry