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Mechanisms involved in the activation of C/EBPα by small activating RNA in hepatocellular carcinoma

Oncogene volume 38, pages 3446–3457 (2019)Cite this article

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Abstract

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPβ occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.

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Acknowledgements

This work was funded by National Taiwan University Hospital and Imperial College London.

Author contributions

VR, NH and KW-H designed the experiments. XYZ performed the experiments. VR and NH supervised the project. VR and XYZ analysed the data, XYZ wrote the paper. VR, NH and KW-H revised the manuscript. JF, HCJ, PS, JR and NH had comments and discussion on the results.

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Authors and Affiliations

  1. HPB Surgery Unit, Hammersmith Hospital, Department of Surgery and Cancer, Imperial College London, London, W12 0HS, UK

    Xiaoyang Zhao, Vikash Reebye & Nagy. A. Habib

  2. Department of Surgery, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Xiaoyang Zhao & Jia Fan

  3. Department of General Surgery, the First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China

    Xiaoyang Zhao & Hongchi Jiang

  4. Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK

    Paul Hitchen

  5. Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, NO-7489, Trondheim, Norway

    Pål Sætrom

  6. Department of Computer Science, Norwegian University of Science and Technology, NO-7489, Trondheim, Norway

    Pål Sætrom

  7. Division of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA

    John Rossi

  8. Department of Surgery and Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan

    Kai-Wen Huang

  9. Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan

    Kai-Wen Huang

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  1. Xiaoyang Zhao
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Correspondence to Kai-Wen Huang.

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VR, PS, JJR and NH own stock in MiNA (Holdings) Limited. All the remaining authors declare that they have no conflict of interest.

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Zhao, X., Reebye, V., Hitchen, P. et al. Mechanisms involved in the activation of C/EBPα by small activating RNA in hepatocellular carcinoma. Oncogene 38, 3446–3457 (2019). https://doi.org/10.1038/s41388-018-0665-6

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