Fig 2: Overexpression of transcription factor HOXB5 reverses the inhibitory effects of ANGPT2 silencing on proliferation of EC-9706 esophageal cancer cells. (A) Relative mRNA and protein expression levels of ANGPT2 were detected using reverse transcription-quantitative PCR and western blot analysis. (B) Viability of EC-9706 cells was detected using Cell Counting Kit-8. **P<0.01 and ***P<0.001 vs. shRNA-NC. ##P<0.01 and ###P<0.001 vs. shRNA-ANGPT2 + pcDNA3.1. (C) Proliferation was detected using EdU staining (scale bar, 50 µm). (D) Colony forming ability was detected using colony formation assay (scale bar, 34 mm). HOXB5, homeobox B5; ANGPT2, angiopoietin-2; shRNA, short harpin RNA; OD, optical density; NC, negative control.

Fig 3: HOXB5 transcription activates ANGPT2. (A) JASPAR-predicted HOXB5 binding motif. (B) HOXB5 was upregulated in patients with esophageal cancer. *P<0.05. (C) HOXB5 upregulation was not significantly associated with low overall survival rate of patients with esophageal cancer. (D) Relative mRNA expression of HOXB5 was detected using RT-qPCR. (E) Protein expression of HOXB5 was detected using western blot analysis. ***P<0.001 vs. HEEC. Relative (F) mRNA and (G) protein expression levels of HOXB5 in HOXB5-overexpressing EC-9706 cells were detected using RT-qPCR and western blot analysis. ***P<0.001 vs. pcDNA3.1. (H) Relative luciferase activity was detected using luciferase reporter assay. ***P<0.001 vs. ANGPT2-WT + pcDNA3.1. (I) Relative enrichment of ANGPT2 was detected using chromatin immunoprecipitation. ***P<0.001 vs. input; ###P<0.001 vs. IgG. HOXB5, homeobox B5; ANGPT2, angiopoietin-2; RT-qPCR, reverse transcription-quantitative PCR; ESCA, esophageal carcinoma; WT, wild type; MUT, mutated; TPM, transcripts per million.

Fig 4: ANGPT2 upregulation in esophageal cancer tissue and cell lines and is associated with poor patient prognosis. (A) ANGPT2 was upregulated in patients with esophageal cancer. *P<0.05 vs. normal. (B) ANGPT2 upregulation was significantly associated with low overall survival rate of patients with esophageal cancer. (C) Relative mRNA expression of ANGPT2 in normal HEEC and esophageal cancer cell lines was detected using reverse transcription-quantitative PCR. (D) Protein expression of ANGPT2 in normal HEEC and esophageal cancer cell lines was detected using western blotting. ***P<0.001 vs. HEEC. ANGPT2, angiopoietin-2; ESCA, esophageal carcinoma; HEEC, human esophageal epithelial cell; TPM, transcripts per million.

Fig 5: ANGPT2 silencing inhibits proliferation of EC-9706 esophageal cancer cells. (A) Relative mRNA expression of ANGPT2 was detected using reverse transcription-quantitative PCR. (B) Protein expression of ANGPT2 was detected using western blot analysis. ***P<0.001 vs. shRNA-NC. (C) Viability of EC-9706 cells was detected using Cell Counting Kit-8. **P<0.01 and ***P<0.001 vs. control. (D) Proliferation of EC-9706 cells was detected using EDU staining assay (scale bar, 50 µm). (E) Colony formation assay of EC-9706 cells (scale bar, 34 mm). ANGPT2, angiopoietin-2; shRNA, short harpin RNA; NC, negative control; OD, optical density.