Journal: Nature Communications
Article Title: HOXA9 inhibits HIF-1α-mediated glycolysis through interacting with CRIP2 to repress cutaneous squamous cell carcinoma development
Figure Lengend Snippet: Loss of HOXA9 leads to enhanced glycolysis and tumor growth in vivo. siNC and siHOXA9 oligos were injected into A431 cell xenografts every 3 days. a Loss of HOXA9 promotes subcutaneous tumor growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to day. Tumor volume statistical data represent the average of four independent experiments ± s.d, respectively. b The mice were sacrificed at the end of the experiment and images taken along with the dissected tumors from five representative mice are shown. White arrows indicate the siNC-treated xenografts whereas black arrows indicate siHOXA9-treated xenografts. Scale bar, 1 cm. c The expression of HOXA9 , HIF1A , HK2 , GLUT1 , and PDK1 was measured in the dissected tumors by qRT-PCR. qRT-PCR statistical data represent the average of four independent experiments ± s.d. d The protein expression of HOXA9, HIF-1α, HK2, GLUT1, and PDK1 was detected in xenografts after siHOXA9 treatment by western blot. e Histopathology analysis (IHC staining) of HOXA9, HIF-1α, HK2, GLUT1, and PDK1 on tumor sections. HOXA9 pre-absorption tests was also performed to validate the specificity of HOXA9 antibody. Scale bar, 100 µm (200×). f Comparison of glucose consumption between siHOXA9-treated and siNC-treated xenograft tumors by microPET/CT imaging of the uptake and retention of 18 F-FDG injected via the tail vein. A representative microPET/CT image is shown. g A model of the miR-365-HOXA9-HIF-1α glycolysis-regulatory axis in cSCC development. In cSCC tumors, loss of HOXA9 up-regulates HIF-1α and its downstream glycolytic genes of HK2 , GLUT1 , and PDK1 in the HIF-1 pathway, which contributes to the enhancement of glycolysis and promotes cSCC progression. In normal skin or HOXA9-treated cSCC, HOXA9 interacts with CRIP2 and epigenetically represses HIF-1α expression, which leads to the replacement of HIF-1α by the HOXA9-CRIP2 complex at the promoter regions and represses the expression of glycolytic genes including HK2 , GLUT1 , and PDK1 , which subsequently contributes to the inhibition of tumor progression. Each experiment was performed in triplicate and data are presented as mean ± s.d. One-Way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (* P
Article Snippet: The following primary antibodies and dilutions were used: HOXA9 (Abcam, ab140631, 1:2000) and CRIP2 (Abcam, ab151496, 1:2000); HIF-1α (Santa Cruz Biotechnology, sc-10790, 1:2000), HK2 (Santa Cruz Biotechnology, sc-374091, 1:2000), GLUT1 (Cell Signaling Technology, 12939S, 1:2000), and PDK1 (Cell Signaling Technology, 3062, 1:2000); and α-TUBULIN (Santa Cruz Biotechnology, sc-5286, 1:2000) and GAPDH (Santa Cruz Biotechnology, sc-25778, 1:5000).
Techniques: In Vivo, Injection, Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Histopathology, Immunohistochemistry, Staining, Imaging, Inhibition