Figure 3 from Hypoxia-Induced Long Noncoding RNA HIF1A-AS2 Regulates Stability of MHC Class I Protein in Head and Neck Cancer

Impact of HIF1A-AS2 on the expression of MHC-Ⅰ in HNSCC. A, CIBERSORT analysis to compare the proportion of infiltrated immune cells in patients from the TCGA-HNSCC database with high HIF1A-AS2 [HIF1A-AS2(H)] versus low HIF1A-AS2 [HIF1A-AS2(L)]. The infiltrated immune cells that significantly decreased in patients with HIF1A-AS2(H) were labeled in green text and conversely were highlighted in red text. B, CIBERSORT analysis to compare the proportion of CD8⁺ T cells in the 4 patient subgroups based on high or low HIF1A and high or low HIF1A-AS2: HIF1A-AS2(H)/HIF1A(H), HIF1A-AS2(H)/HIF1A(L), HIF1A-AS2(L)/HIF1A(H), and HIF1A-AS2(L)/HIF1A(L). C, Top: western blots showing the expression of HIF1α and HLA-ABC in SAS (left) and HSC3 (right) cells treated with CoCl₂ treated versus corresponding control (Ctrl) and overexpressing HIF1A-AS2 versus control (Vec). Bottom: RT-qPCR for showing the HIF1A-AS expression in the above groups of cells. Data represent the mean ± S.D. n = 3 independent experiments (each experiment contained two technical replicates). D, Top: Representative flow cytometry overlay histograms showing the cell surface expression of HLA-ABC in SAS and HSC3 cells overexpressing HIF1A-AS2 and control cells. Bottom: mean fluorescent intensity quantification was performed for independent replicate experiments (n = 3). E, Top: western blots of the expression of HLA-ABC in SAS HIF1A-AS2 knockdown cells (SAS-sh#1 and SAS-sh#2) under hypoxia-mimic versus normoxia condition. Lower: RT-qPCR for examination of HIF1A-AS2 level in SAS-sh#1 and SAS-sh#2 cells under hypoxia-mimic versus normoxia condition. #1 and #2 represent two different shRNAs. Data represent the mean ± SD. n = 3 independent experiments (each experiment contained two technical replicates). F, Western blots for showing the expression of HLA-ABC in SAS HIF1A-AS2 knockdown cells (SAS-sh#1) and transiently reconstituted with HIF1A-AS2 versus control under hypoxia-mimic conditions. *, P < 0.05; **, P < 0.01; ***, P < 0.001.