Fig 2: miR-205 targeted regulation of HOXD9. (A) Three major databases were used to predict target genes of miR-205. Based on the final biological function, HOXD9 was selected. (B) The correlation between miR-205 and HOXD9 mRNA expression was calculated in 100 pairs of TNBC specimens. (C) The correlation between miR-205 and HOXD9 protein expression was calculated in 100 pairs of TNBC specimens. (D) HOXD9 protein expression in miR-205 overexpression treated MDA-MB-231 cells was lower than that in normal MDA-MB-231 cells. (E) Western blot analysis of the HOXD9 protein and mRNA expression in miR-205 knockdown treated BT-549 cells and miR-205 overexpression treated MDA-MB-231 cells. (F) qRT-PCR analysis of the HOXD9 protein and mRNA expression in miR-205 knockdown treated BT-549 cells and miR-205 overexpression treated MDA-MB-231 cells. (G) Construction of HOXD9 mRNA 3’UTR wild-type and mutant plasmids using potential binding sites of miR-205 and HOXD9 mRNA. (H) Dual luciferase experiment was used to verify the binding of miR-205 and HOXD9 mRNA. (I) Western blot analysis of HOXD9 siRNA. (J) qRT-PCR analysis of HOXD9 siRNA. (K) Cell proliferation analysis of the miR-205 and HOXD9 knockdown treated BT-549 cells after different incubation time. (L) Cell proliferation analysis of the miR-205 and HOXD9 knockdown treated BT-549 cells with Cisplatin treatment. (M) Cell proliferation analysis of the miR-205 and HOXD9 knockdown treated BT-549 cells with Doxorubicin treatment. (N) Cell proliferation analysis of the miR-205 and HOXD9 knockdown treated BT-549 cells with Paclitaxel treatment. ***P<0.001.

Fig 3: HOXD9 regulates glioma cells proliferation, apoptosis, glucose uptake and aerobic glycolysis.HOXD9 expression in U87 cells transfected with the vector or HOXD9 overexpression. A The mRNA was quantified. B The protein was quantified. **p < 0.01, ***p < 0.001. C qRT-PCR detected the mRNA expression of PLEKHA4, ***p < 0.001. D Western Blot detected the protein expression of PLEKHA4, ***p < 0.001. E Luciferase reporter assay shows that HOXD9 binds to PLEKHA4, ***p < 0.001. The luciferase reporter plasmid containing WT or MUT-PLEKHA4 was co-transfected into U87 cells with HOXD9 overexpression or vector. F The mRNA, and G, H. The protein expression levels of HOXD9 and PLEKHA4 in U87 cells co-transfected with shPLEKHA4 and HOXD9 overexpression. ***p < 0.001 compared with Vector + shNC, #p < 0.05, ###p < 0.001 compared with Vector + shPLEKHA4 group. I CCK-8 detected the activity of the U87 cells. ***p < 0.001 compared with Vector + shNC group, ###p < 0.001 compared with Vector + shPLEKHA4 group. J, K Flow cytometry detected apoptosis. ***p < 0.001 compared with Vector + shNC group, ###p < 0.001 compared with Vector + shPLEKHA4 group. L Glucose uptake assays, M lactate production, N ATP production were measured, ***p < 0.001 compared with Vector + shNC group, ##p < 0.01, ###p < 0.001 compared with Vector + shPLEKHA4 group. The cell experiments were conducted three independent cultures, with two replicates conducted each time.