Genome-wide CRISPR knockout screens identify ADAMTSL3 and PTEN genes as suppressors of HCC proliferation and metastasis, respectively.

PURPOSE:It is important for hepatocellular carcinoma (HCC) treatment that the targets related to its progression are identified. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9)-based genetic screening is a powerful tool for identifying genes with loss-of-function mutations that are critical for tumour growth and metastasis. METHODS:We transduced the human SMMC7721 HCC cell line expressing Cas9 with a human genome-scale CRISPR-Cas9 knockout (GeCKO) lentiviral library A (hGeCKOa) of 65,383 single-guide RNAs (sgRNAs) targeting 19,050 human genes; we then subcutaneously transplanted the transduced cells into nude mice. RESULTS:The transduced cells were found to proliferate and metastasize faster than the untransduced cells. Through next-generation sequencing, the genes potentially related to HCC proliferation and metastasis were identified. The sgRNAs targeting the ADAMTSL3 and PTEN genes appeared twice on the list of genes related to HCC proliferation and metastasis, respectively. Analysis based on the data mining of Oncomine revealed that the ADAMTSL3 and PTEN genes were expressed at lower levels in HCC cells than they were in normal liver cells, indicating their tumour-suppressive roles. Downregulation of ADAMTSL3 and PTEN displayed poor overall survival (OS) and predicted poor relapse-free survival (RFS), further supporting their tumour-suppressive roles. Moreover, knocking out either the ADAMTSL3 or PTEN genes promoted either the proliferation or metastasis of HCC cells, respectively. CONCLUSIONS:Using both in vitro and in vivo approaches, we described the profound role of the ADAMTSL3 and PTEN genes. This study indicates novel candidate targets for use in HCC treatment and therapy.

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