Figure 2: LncHIFCAR functions as an oncogene driving oral cancer progression.

(a) Expression levels of LncHIFCAR in oral cancer cell lines. The levels of LncHIFCAR were normalized against RPLP0 mRNA level and are presented relative to the expression in OEC-M1. (b) Establishment of LncHIFCAR-knockdown SAS clones. Upper panel: schematic representation of the genomic structure of LncHIFCAR/MIR31HG. The targeting sites of the designed siRNAs are indicated by gray arrowhead. LncHIFCAR/MIR31HG is composed of four exons (filled blue boxes) and the miR-31, located within intron 1, is shown with a red horizontal bar. Lower panel: LncHIFCAR and miR-31 expression levels in siRNA-transfected SAS cells were analysed by qPCR and normalized to GAPDH and U6 level, respectively. (c) LncHIFCAR promotes oral cancer cell invasion and migration. Transwell invasion and migration assays of siRNA-transfected SAS cells were performed. Representative photos and quantitative analysis are shown. Scale bar, 100 μm. (d) Cell growth curve of the siRNA-transfected SAS cells under normoxia or hypoxia. (e,f) Knockdown of LncHIFCAR reduces hypoxia-induced glucose uptake (e) and lactate production (f). The glucose and lactate levels in the culture media of the siRNA-transfected SAS cells were measured after 16 h of normoxia or hypoxia treatment. The data are presented as fold difference compared with the level of negative control siRNA-transfected cells in normoxia. (g) Knockdown of LncHIFCAR reduces the sphere-forming ability of SAS cells. The siRNA-transfected SAS cells were grown in suspension culture to form sphere. Representative phase-contrast microscopic images of the cell aggregates are displayed (scale bar, 300 μm). Bar graph represents the total number of spheres or the relative number of spheres with diameter >1 mm. Graphs show mean±s.d. NC, negative control. n, the number of independent experiments performed; Student’s t-test, *P<0.05, **P<0.01 and ***P<0.001.