Fig. 1: HOXA10-HOXA9 encodes a microprotein HDSP in GC.

a Analyzing GC datasets from TCGA, GC-related datasets from GEO, and ribosome sequencing datasets, the differentially expressed lncRNAs in various datasets were intersected to create a Venn diagram. b Polysome-bound RNA isolation and qRT-PCR were used to analyze the enrichment of the five candidate lncRNAs on polyribosomes in SGC-7901 cells. Mean ± SD, n = 3 biologically independent experiments. c QRT-PCR analysis of HOXA10-HOXA9 expression in 70 paired GC tissues. Mean ± SD, Paired samples, 2-sided Student’s t test. ****P < 0.0001. d QRT-PCR analysis of HOXA10-HOXA9 expression levels in 7 GC cell lines and normal gastric mucosal epithelial cells (GES-1). Mean ± SD, Unpaired two‐tailed Student’s t test. n = 3 biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e A schematic diagram shows the HOXA10-HOXA9 ORF2 encoding a 112aa small protein. f Schematic representation of the structures of HDSP-GFP, HDSP-Flag, and their respective mutant variants. g The expression vector depicted in (f) was separately transfected into GC cells. After 48 h, the expression of the HDSP-GFP fusion protein (left) and HDSP-Flag fusion protein (right) were assessed using western blot analysis. The experiment was repeated 3 times independently with similar results. h Following transfection of GFP, HDSP-GFP, and their respective mutant plasmids into GC cells, the green fluorescence emitted by GFP was visualized using a fluorescence microscope. The experiment was repeated 3 times independently with similar results. i After transfecting GC cells with HDSP-Flag and its mutant vector, the subcellular localization of the fusion protein was examined using immunofluorescence. The experiment was repeated 3 times independently with similar results. j The sequences of HOXA10-HOXA9 were cloned into overexpression vectors containing T7 promoters, and their coding potential was validated through in vitro translation experiments. The experiment was repeated 3 times independently with similar results. k The schematic diagram illustrates the Flag Knock-In Strategy. The insertion of the Flag tag after the start codon of HDSP represents a fusion expression. l Western blot was employed to ascertain the successful integration of the Flag tag into GC cells. The experiment was repeated 3 times independently with similar results. m After introducing Flag via knock-in in GC cells, HOXA9 was subsequently knocked out, followed by the assessment of HOXA9 and HDSP expression levels using western blot analysis. The experiment was repeated 3 times independently with similar results. Source data are provided as a Source data file.