Fig 2: Modulation of RBM8A expression affects the migratory and invasive potential of parental cell lines (PCLs) and drug-resistant (DR)-hepatocellular carcinoma (HCC) cells, as well as the expression of proteins related to drug resistance. (A) Wound-healing assay. The scraped areas were photographed at 0, 24, and 72 h after scraping. Migration efficiency was quantitated at 24 and 72 h after scraping (right panel). Magnification, 10×. Scale bar, 200 µm. (B) Transwell assay. Representative examples of each experimental group are shown. Migration efficiency was quantitated at 24 and 72 h (right panel). Magnification, 40×. Scale bar, 50 µm. (C) Matrigel-Transwell assay. Representative photographs and quantitation were shown. Data were either representative of three similar observations, or were shown as the mean ± SD of three experiments. Magnification, 40×. Scale bar, 50 µm. (D) Western blot analysis of PCL-Bel7404-NC, PCL-Bel7404-RBM8A-KD, DR-Bel7404-NC and DR-Bel7404-RBM8A-KD. Cells were analyzed without OXA treatment (second row) or with OXA treatment (third row). Data were representative of three similar observations or were shown as the mean ± SD of three experiments. (E) Western blot analysis of PCL-MHCC97H-NC, PCL-MHCC97H-RBM8A-OE, DR-MHCC97H-NC and DR-MHCC97H- RBM8A-OE cells as in (D).

Fig 3: Molecular network showing how RBM8A may regulate oxaliplatin (OXA) resistance in hepatocellular carcinoma (HCC). (A) Bioinformatics analysis integrating the regulatory information of RBM8A on module genes and pivot factors to construct a comprehensive overview of RBM8A-mediated OXA resistance in HCC. In this landscape, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and transcription factors (TFs) mediate the ability of RBM8A-regulated module genes and their downstream signaling pathways to confer drug resistance on HCC cells. (B) Western blot analysis of the expression of transcription factors MYC, STAT3, P53, E2F1, YY1, HDAC1, and HDAC9 in HCC cell lines. Western blotting revealed that, after overexpression or knockdown of RBM8A in parental cell lines (PCLs) and drug-resistant (DR)-HCC cells, HDAC9 expression regulated by RBM8A was associated with OXA resistance in HCC cells. (C) Bioinformatics analysis combined with quantitative real time PCR (qRT-PCR) and western blotting revealed that HDAC9 is the pivotal transcription factor most closely related to the RBM8A-mediated regulation of OXA resistance in HCC. The HDAC9-module gene-KEGG signaling pathway was extracted, and the potential mechanism by which the RBM8A-HDAC9 axis regulates drug resistance in HCC was identified.

Fig 4: Effects of RBM8A on drug-resistant (DR)-hepatocellular carcinoma (HCC) tumorigenesis in vivo. (A) Bel7404/OXA-RBM8A-KD and control cells were injected orthotopically into mammary fat pads of nude mice, which were then injected with OXA at 10 mg/kg around the tumor at 1, 2, 4, and 6 weeks. The growth of tumors was followed during a six-week period. Photographs of primary tumors are shown on the right. (B) Comparison of tumor volume in Bel7404/OXA-RBM8A-KD and Bel7404/OXA-RBM8A-NC animals. Mice injected with Bel7404/OXA-RBM8A-KD cells formed smaller (p < 0.0001) and lighter (p = 0.0004) tumors than mice injected with control cells (NC). ***<0.001 (C, D) Immunohistochemical staining and western blotting of Bel7404/OXA-RBM8A-KD and Bel7404/OXA-RBM8A-NC tumors. Data were expressed as the mean ± SD of three independent experiments or were representative of three independent observations. Magnification, 20×. Scale bar, 100 µm.

Fig 5: Involvement of the epithelial–mesenchymal transition (EMT) in RBM8A-mediated proliferation, invasion and drug resistance of hepatocellular carcinoma (HCC) cells. (A) Cell proliferation was analyzed in PCL-MHCC97H-NC, PCL-MHCC97H-RBM8A-OE, DR-MHCC97H-NC and DR-MHCC97H- RBM8A-OE cells in the presence or absence of the EMT inhibitor C19 using the CCK8 assay. (B) Wound-healing assay with or without EMT inhibitor C19. The scraped areas were photographed at 0 and 48 h after scraping. Migration efficiency was quantitated at 48 h after scraping (right). (C) Transwell analysis with or without EMT inhibitor C19. (D) Matrigel-Transwell analysis with or without EMT inhibitor C19. Magnification, 40×. Scale bar, 50 µm.