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In vivo genome-wide CRISPR activation screening identifies functionally important long non-coding RNAs in hepatocellular carcinoma

  • Author Footnotes
    ∗ Authors contributed equally to this work.
    Lok-Sze Wong
    Footnotes
    ∗ Authors contributed equally to this work.
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Author Footnotes
    ∗ Authors contributed equally to this work.
    Lai Wei
    Footnotes
    ∗ Authors contributed equally to this work.
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Geng-chao Wang
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Cheuk-Ting Law
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Ho-Ching Tsang
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Wai-Ching Chin
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Irene OL. Ng
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Chun-Ming Wong
    Correspondence
    Corresponding author: Dr. Chun-Ming Wong, State Key Laboratory of Liver Research and Department of Pathology, Rm 701, 7/F, Hong Kong Jockey Club of Inter-disciplinary Research, 5 Sassoon Road, The University of Hong Kong, Pokfulam, Hong Kong
    Affiliations
    The State Key Laboratory of Liver Research, Department of Pathology, Li-Ka Shing Faculty of Medicine, The University of Hong Kong
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  • Author Footnotes
    ∗ Authors contributed equally to this work.
Open AccessPublished:August 06, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.07.017
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      Abstract

      Background and Aims

      Long non-coding RNAs (lncRNAs) are found to have profound impacts on diverse cellular processes. Although high-throughput sequencing studies have revealed the differential lncRNA expression profiles between hepatocellular carcinoma (HCC) and non-tumor livers, the functional impacts of lncRNAs on HCC development await further investigation. Herein, we sought to address the functional roles of lncRNAs in HCC pathogenesis by in vivo functional screening.

      Methods

      We performed genome-wide CRISPR/dCas9 lncRNA activation screening in HCC xenografts. We characterized the clinical relevance of positively selected lncRNAs using transcriptomic datasets. We employed CRISPR-based gene activation and knockdown approaches to demonstrate the functional roles of positively selected lncRNAs including CASC11 in HCC. RNA-Seq and ChIRP-Seq were used to investigate the molecular mechanisms of CASC11 in HCC progression.

      Results

      The in vivo functional screening identified 1603 positively selected lncRNAs, and 538 of which were overexpressed in HCC patients. Systematic transcriptomic data analysis and clinical investigation revealed that patients with high expression of these lncRNA candidates correlated with aggressive tumor behaviours. Overexpression of these lncRNAs aggravated HCC cell growth. Detailed characterization of a lncRNA candidate, CASC11, demonstrated its pivotal role in cell proliferation and tumor growth. Mechanistically, ChIRP-Seq revealed that CASC11 was bound to the CASC11/MYC shared promoter region on chromosome 8q24. CASC11 modulated the transcriptional activity of MYC in a cis-regulatory manner, which affected the expression of MYC downstream target genes, consequently promoting G1/S progression.

      Conclusions

      Our study demonstrated the power of in vivo CRISPR screening which comprehensively interrogated the functionality of lncRNAs in HCC progression, providing a rationale for targeting these lncRNAs clinically.

      Keywords