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. 2023 May 24;14(5):341. doi: 10.1038/s41419-023-05862-5

Fig. 1. Homeobox D9 (HOXD9) represses transcription of PAX-interacting protein 1-antisense RNA 1 (PAXIP-AS1).

Fig. 1

A Whole lysates of human gastric cancer (GC) and normal mucous cell lines were collected, and HOXD9 expression was detected by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. B Total RNA was extracted from LV-HOXD9 and LV-Vector MKN-74 cells and subjected to RNA sequencing analysis. The colour scale illustrates the relative expression of lncRNAs. C1, C2 Vector and HOXD9 plasmid (C1) or Scr siRNA and HOXD9 siRNAp (C2) were transfected into GC cells. The expression of HOXD9 and PAXIP1-AS1 was detected in GC cells using qPCR. ****P < 0.001, Vector vs. HOXD9; ***P < 0.01 and ****P < 0.001, Scr siRNA vs. HOXD9 siRNAp. Scr siRNA, scrambled siRNA; siRNAp, siRNApool; siRNAp: siRNA1~ siRNA3, siRNAs: final concentration = 33.3%. D Transcriptional factor HOXD9 binding motif was predicted by Jaspar software. E Schematic diagram of PAXIP1-AS1 promoter with two potential HOXD9-binding sites and corresponding mutant binding sites. F ChIP assay demonstrated the direct binding of HOXD9 to the PAXIP1-AS1 promoter in AGS and MKN-74 cells. G Luciferase reporter assay showing the transcriptional regulation ability of HOXD9 to the PAXIP1-AS1 promoter. Luciferase constructs containing PAXIP1-AS1 full-length or mutant promoter were co-transfected with the HOXD9 or Vector plasmid into AGS and MKN-74 cells, and the luciferase activity was measured. Selective mutagenesis of −1503 to −1513 bp from the transcription start site resulted in a loss of response to HOXD9, indicating that this region was HOXD9-responsive (n = 3). *P > 0.05 and ****P < 0.001, Vector vs. HOXD9. WT, wild type and MUT, mutated.