Fig 2: HOXA11-AS1 knockdown inhibited immune escape and metastasis by regulating PD-L1 and downregulating FOSL1 in vivo. FaDu and Detroit cells stably expressing shNC, shHOXA11-AS1-1, or shHOXA11-AS1-2 were injected subcutaneously into NOD-SCID mice to establish xenograft models. PBS or PBMCs were intraperitoneally injected into NOD-SCID mice to kill the xenografts more effectively. (A) HE, Ki67, and IHC staining were used to measure the tumor pathology, proliferation, and PD-L1 expression of xenografts treated with or without PBMCs or PBC. (B) The percentage of Ki67 cells was evaluated in xenografts. (C) The cytotoxic effect of treatment with or without PBMCs or PBS was measured by calculating xenograft volumes in shNC, shHOXA11-AS1-1, and shHOXA11-AS1-2 xenografts, and results are shown in the bar chart as tumor regression rate. (D–F) Tumor volume and weight were detected after HOXA11-AS1 knockdown in vivo. (G) RT-qPCR was used to detect HOXA11-AS levels, and Western blot was used to measure the expression of PD-L1, FOSL1, and PTBP1 after HOXA11-AS1 knockdown. (H,I) The ability of metastasis to the lung, tumor pathology, and nodule numbers of lung metastases were measured after of HOXA11-AS1 knockdown. * p < 0.05, ** p < 0.01, *** p < 0.001. Original Blots see Supplementary File S1.

Fig 3: HOXA11-AS1 promoted PD-L1 expression by upregulating FOSL1 levels through PTBP1, thereby facilitating immune escape, growth, and metastasis of HSCC cells. FaDu and Detroit cells were transfected with pcDNA3.1-HOXA11-AS1 (HOXA11-AS1), pcDNA3.1-PTBP1 (PTBP1), sh-PTBP1, sh-PD-L1, or a combination of HOXA11-AS1 + sh-PTBP1 and HOXA11-AS1 + sh-PD-L1: (A) HOXA11-AS1, FOSL1, and PD-L1 levels were measured in FaDu and Detroit 562 cells treated with HOXA11-AS1 plasmid. (B) Relative expression of PTBP1, FOSL1, and PD-L1 was evaluated after overexpressing PTBP1. (C) Relative levels of PTBP1, FOSL1, and PD-L1 in HOXA11-AS1, shPTBP1, or HOXA11-AS1 + shPTBP1 groups. FaDu and Detroit cells were transfected with HOXA11-AS1, shPD-L1, or a combination of HOXA11-AS1 + shPD-L1: (D) Relative expression of PTBP1, FOSL1, and PD-L1 in cells transfected with HOXA11-AS1, sh-PD-L1, or HOXA11-AS1 + sh-PD-L1. Then, (E) the percentage of CD8+ and CD4+ T cells and (F) the concentration of IFN-? were measured by flow cytometry and ELISA, respectively, and (G–J) the viability, colony formation, migration, and invasion of treated HSCC cells were analyzed by CCK-8, colony formation, wound healing, and transwell assays. Representative images (200×) are shown. * p < 0.05, ** p < 0.01, *** p < 0.001. Original Blots see Supplementary File Figure S1.

Fig 4: NONHSAT024276 directly interacted with polypyrimidine tract–binding protein 1 (PTBP1). A, Flowchart depicting the identification of NONHSAT024276-binding proteins via RNA pull-down, silver staining, mass spectrometry (MS), and Western blot assays. B, Precipitated proteins from RNA pull-down assay were resolved using SDS-PAGE followed by silver staining. C, Statistics of NONHSAT024276-binding proteins identified in HCCLM3 and Mahlavu cells. Top, Venn diagram showing the overlap of RNA-binding proteins (RBPs) identified in HCCLM3 and Mahlavu cells. Middle, enrichment degree of 307 RBPs in both HCCLM3 and Mahlavu cells. Bottom, statistics of RBPs in HCCLM3 and Mahlavu cells. D, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results of RBPs identified in HCCLM3 (left) and Mahlavu (right) cells. E, F, Gene Ontology (GO) analysis results of RBPs identified in HCCLM3 (E) and Mahlavu (F) cells. G, Venn diagram showing overlapping molecules in catRAPID-predicted proteins, differentially expressed mRNAs of microarray, and RNA pull-down proteins. H, Western blot analysis of the proteins retrieved from RNA pull-down assays. I, The interaction between PTBP1 and NONHSAT024276 was verified via RNA immunoprecipitation (RIP) assays. After transfection using ov-276 or ov-NC in HCCLM3 and Mahlavu cells, the cell lysates were incubated with PTBP1 antibody, and the relative fold enrichment of NONHSAT024276 was determined via qRT-PCR compared with input control. J-L, The catRAPID was used to predict the interaction region between NONHSAT024276 and PTBP1

Fig 5: NONHSAT024276 repressed hepatocellular carcinoma (HCC) proliferation and glycolysis via the polypyrimidine tract–binding protein 1–pyruvate kinase (PTBP1-PKM) axis. A, PTBP1 and PKM1/PKM2 ratio downregulation were essential for NONHSAT024276-mediated proliferation inhibition. B, D, Detection of glucose consumption (B), lactate production (C), and ATP production (D) showed that the transfection of ov-PTBP1 or ov-PKM2 plasmid abolished the NONHSAT024276-mediated glycolysis inhibition. E, Expression of PTBP1 and PKM2 was analyzed using Western blot. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001