Journal: Molecular Cancer

Article Title: A novel IFNα-induced long noncoding RNA negatively regulates immunosuppression by interrupting H3K27 acetylation in head and neck squamous cell carcinoma

doi: 10.1186/s12943-019-1123-y

Figure Lengend Snippet: LncMX1–215 negatively regulates IFNα-induced PD-L1 and galectin-9 expression. a lncMX1–215 expression was detected in HN4 and Cal27 cells via PCR after transfection with expression plasmids for 24 h. b, c The protein and mRNA of PD-L1 and galectin-9 expression levels were detected after transfection with lncMX1–215 for 24 h followed by 200 ng/ml IFNα stimulation for 24 h. d The lncMX1–215 knockdown efficiency after transfection with ASO for 24 h was analyzed. e, f After ASO transfection for 24 h followed by 200 ng/ml IFNα stimulation for 24 h, PD-L1 and galectin-9 expression levels were detected using western blotting and qRT-PCR. g Surface PD-L1 expression was examined using flow cytometry after transfection with lncMX1–215 followed by 200 ng/ml IFNα stimulation for 24 h. h The galectin-9 concentration in medium supernatant was measured via ELISA after transfection for 24 h followed by 200 ng/ml IFNα stimulation for 24 h. i Galectin-9 knockdown efficiency was detected via western blotting after transfection with siRNA for 48 h. j Transfected cells were seeded in a 96-well plate and incubated with NK cells for 4 h at various effector/target (E:T) cell ratios as indicated. The specific lysis rate was measured using an LDH kit. k Relative CD274 mRNA and lncMX1–215 expression were detected using qRT-PCR after 200 ng/ml IFNα stimulation for indicated time. *: P < 0.05; **: P < 0.01

Article Snippet: IFNα and recombinant human galectin-9 were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Lysis

Journal: Molecular Cancer

Article Title: A novel IFNα-induced long noncoding RNA negatively regulates immunosuppression by interrupting H3K27 acetylation in head and neck squamous cell carcinoma

doi: 10.1186/s12943-019-1123-y

Figure Lengend Snippet: Gain of H3K27 acetylation activates PD-L1 and galectin-9 expression. a Layered H3K27 acetylation enrichment in the upstream region of PD-L1 and LGALS9, as shown in the UCSC database. b PD-L1 and acetylation of histone 3 were detected and quantified after treatment with 200 ng/ml IFNα or 5 μM MS-275 for 24 h. # indicated the deference between combined group and each alone. c PD-L1, H3K27ac, H3K18ac and H3K9ac were detected and quantified after the indicated MS-275 treatment for 24 h. d PD-L1 and H3K27ac were detected and quantified after 5 μM MS-275 treatment for the indicated time. e After treatment of HN4 and Cal27 cells with 15 μM SAHA or 5 μM MS-275 for 24 h, ChIP assays were performed using anti-H3K27ac antibody. f The promoter activity of PD-L1 and LGALS9 was measured after transfection with lncMX1–215 for 24 h and then SAHA or MS-275 treatment for 24 h in 293 T cells. g, h The relative mRNA of CD274 and LGALS9 were detected after transfection with lncMX1–215 and then 15 μM SAHA or 5 μM MS-275 treatment for 24 h in HN4 and Cal27 cells. *: P < 0.05; **: P < 0.01

Article Snippet: IFNα and recombinant human galectin-9 were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Expressing, Activity Assay, Transfection

Journal: Molecular Cancer

Article Title: A novel IFNα-induced long noncoding RNA negatively regulates immunosuppression by interrupting H3K27 acetylation in head and neck squamous cell carcinoma

doi: 10.1186/s12943-019-1123-y

Figure Lengend Snippet: LncMX1–215 directly interacts with GCN5. a GCN5 and H3K27ac were detected after transfection with lncMX1–215 for 24 h and treatment with 200 ng/ml IFNα for 24 h. b GCN5 and H3K27ac were detected after transfection with ASO for 48 h. c, d GCN5, PD-L1, galectin-9 and H3K27ac were examined after transfection with lncMX1–215 or ASO for 24 h followed by treatment with 5 μM MS-275 for 24 h. e GCN5, PD-L1, galectin-9 and H3K27ac were detected after ectopic expression of GCN5 for 48 h in HN4 and Cal27 cells. f PD-L1 and galectin-9 were detected after cotransfection with GCN5 and lncMX1–215 for 48 h. g After transfection with lncMX1–215 for 48 h, cell lysates were precipitated with anti-GCN5 or IgG antibody. h GCN5 and H3K27ac expression was analyzed and quantified using immunofluorescence. i The correlation between H3K27ac and GCN5 and lncMX1–215 expression was analyzed in HNSCC tissue microarray. j Overexpression of GCN5-DYKDDDDK fusion plasmid in HN4 cells was verified by western blotting. P1 to P5 were truncations for full length of lncMX1–215 amplified by specific primers. k After transfection of HN4 cells with GCN5-DYKDDDDK fusion plasmid for 48 h, RNA immunoprecipitation was used to detect binding of GCN5 and lncMX1–215. The binding fragments were detected using PCR with primers P1 to P5. l Cell lysates were incubated with biotinylated lncMX1–215 or an unrelated probe, and the eluent and flow-through were analyzed via western blotting with anti-GCN5 antibody. L: lysate load; FT: flow-through; E: eluent. *: P < 0.05; **: P < 0.01

Article Snippet: IFNα and recombinant human galectin-9 were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Transfection, Expressing, Cotransfection, Immunofluorescence, Microarray, Over Expression, Plasmid Preparation, Western Blot, Amplification, Immunoprecipitation, Binding Assay, Incubation

Journal: Molecular Cancer

Article Title: A novel IFNα-induced long noncoding RNA negatively regulates immunosuppression by interrupting H3K27 acetylation in head and neck squamous cell carcinoma

doi: 10.1186/s12943-019-1123-y

Figure Lengend Snippet: A negative correlation between lncMX1–215 and PD-L1 and galectin-9 expression was observed in HNSCC tissues. a Representative images of lncMX1–215 expression in normal and HNSCC tissues shown using the RNAscope technique. b The expression scores of lncMX1–215 in normal and HNSCC tissues were compared. c-f, Correlations between lncMX1–215 expression and TNM stage, pathological grade, gender and age were analyzed. g Representative images of PD-L1 and lncMX1–215 expression were shown. h The correlation between PD-L1 and lncMX1–215 expression was analyzed in an HNSCC TMA. i Representative images of galectin-9 and lncMX1–215 expression were shown. j The correlation between galectin-9 and lncMX1–215 expression was analyzed in an HNSCC TMA. The red triangle indicates an lncMX1–215 molecule. *: P < 0.05; **: P < 0.01

Article Snippet: IFNα and recombinant human galectin-9 were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: A novel IFNα-induced long noncoding RNA negatively regulates immunosuppression by interrupting H3K27 acetylation in head and neck squamous cell carcinoma

doi: 10.1186/s12943-019-1123-y

Figure Lengend Snippet: Overexpression of lncMX1–215 suppresses HNSCC metastasis. a, b Migration and invasion assays were performed with transfected cells using Transwell inserts (bar = 100 μm, 20 μm, respectively). c snail was detected in HN4 and Cal27 cells after transfection with lncMX1–215 or ASO for 48 h. d Representative images of lungs from mice in each experimental group. The arrows indicate individual metastatic nodules. Metastatic tumors in lung tissues were identified via hematoxylin and eosin staining. e The number of metastatic nodules in each group was analyzed. f Schematic diagram showing that IFNα-induced lncMX1–215 can negatively regulate PD-L1 and galectin-9 expression by interrupting GCN5/H3K27ac binding in HNSCC. *: P < 0.05; **: P < 0.01

Article Snippet: IFNα and recombinant human galectin-9 were purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Over Expression, Migration, Transfection, Staining, Expressing, Binding Assay