Mechanism and Molecular Network of RBM8A-Mediated Regulation of Oxaliplatin Resistance in Hepatocellular Carcinoma

Abstract

RNA-binding motif protein 8A (RBM8A) is abnormally overexpressed in hepatocellular carcinoma (HCC) and involved in the epithelial-mesenchymal transition (EMT). The EMT plays an important role in the development of drug resistance, suggesting that RBM8A may be involved in the regulation of oxaliplatin (OXA) resistance in HCC. Here we examined the potential involvement of RBM8A and its downstream pathways in OXA resistance using in vitro and in vivo models. RBM8A overexpression induced the EMT in OXA-resistant HCC cells, altering cell proliferation, apoptosis, migration, and invasion. Moreover, whole-genome microarrays combined with bioinformatics analysis revealed that RBM8A has a wide range of transcriptional regulatory capabilities in OXA-resistant HCC, including the ability to regulate several important tumor-related signaling pathways. In particular, histone deacetylase 9 (HDAC9) emerged as an important mediator of RBM8A activity related to OXA resistance. These data suggest that RBM8A and its related regulatory pathways represent potential markers of OXA resistance and therapeutic targets in HCC.

Keywords: RNA-binding motif protein 8A; drug resistance; hepatocellular carcinoma; histone deacetylase 9; molecular network; oxaliplatin.

Figure 1

Selection of OXA-resistant hepatocellular carcinoma (HCC) cells and establishment of cell lines in which RBM8A was overexpressed or knocked down. (A) Real time (RT)-PCR and western blot analysis of RBM8A expression in HCC cell lines. Western blot results were quantitated. (B) Schematic representation of the protocol used to obtain OXA-resistant HCC cells from the parental cell line (PCL). During concentration-elevation and intermittent induction treatment with OXA, each dose was maintained for 15 days. OXA-resistant cell lines were obtained by the end of 6 months. (C) Representative phase contrast images of Bel7404 PCLs and drug-resistant cells (DR-HCC cells, left panels) or MHCC97H PCLs and DR-HCC cells (right panels). Magnification, 20×. Scale bar, 20 μm. (D) Knockdown (KD) and overexpression (OE) efficiency of RBM8A in PCLs and DR-HCC cells based on RT-PCR and western blot analysis, compared with the negative control (NC). Western blot data were quantitated (right panels). Data were expressed as mean ± SD of three independent experiments, or were representative of three independent observations.

Figure 2

Modulation of RBM8A expression affects proliferation, apoptosis and cell cycle progression in parental cell lines (PCLs) and drug-resistant (DR)-hepatocellular carcinoma (HCC) cells. (A) Cell proliferation measured using the Cell Counting Kit-8. *P<0.001. (B) Half maximal inhibitory concentration (IC₅₀) of oxaliplatin (OXA) when cells were treated for 48 h. (C) Apoptosis determined by flow cytometry. Representative quadrant figures were presented on the left, and rates of apoptotic PCLs and DR-HCC cells were shown on the right. (D) Apoptosis in PCLs and DR-HCC cells at 48 h after OXA treatment.

Figure 3

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).

Figure 4

Modulation of RBM8A expression affects the epithelial–mesenchymal transition (EMT) in parental cell lines (PCL) and drug-resistant (DR)-hepatocellular carcinoma (HCC) cells. (A–D) Immunofluorescence staining of the (A) cytoskeleton, (B) E-cadherin, (C) N-cadherin, and (D) Snail (all red). All confocal microscopy images show the merging with DAPI (blue) in PCLs and DR-HCC cells upon RBM8A knockdown or overexpression. Scale bar, 20 μm. (E) Western blot analysis of E-cadherin, N-cadherin, and Snail in PCL-Bel7404 and DR-Bel7404 cells with or without RBM8A knockdown. Cells were analyzed without OXA treatment (second row) or with OXA treatment (third row). Data were expressed as the mean ± SD of three independent experiments or were representative of three independent observations. (F) Western blot analysis of E-cadherin, N-cadherin, and Snail protein expression in PCL-MHCC97H and DR-MHCC97H cells with or without RBM8A overexpression as in (E).

Figure 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.

Figure 6

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.

Figure 7

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.