Abstract
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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Data availability
The data that support the findings of this study are available from the corresponding authors upon reasonable request.
Abbreviations
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- PBS:
-
Phosphate-buffered saline
- HCC:
-
Hepatocellular carcinoma
- DENA:
-
Diethylnitrosamine
- GeCKO:
-
Genome-scale CRISPR- Cas9 knockout
- sgRNAs:
-
Single-guide RNAs
- CRISPR-Cas9:
-
Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9
- FBS:
-
Fetal bovine serum
- MOI:
-
Multiplicity of infection
- DAB:
-
3, 3′- Diaminobenzidine
- EMT:
-
Epithelial–mesenchymal transition
- BSD:
-
Blasticidin
- H&E staining:
-
Hematoxylin–eosin staining
- PAM:
-
Protospacer adjacent motif
- CCK-8:
-
Cell counting kit-8
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Acknowledgements
We thank the Tianjin Medical University Cancer Hospital for the assistance with the HCC and adjacent tissue patient biopsies used in this study.
Funding
This study was funded by the National Natural Science Foundation of China (No. 81501531).
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XLZ and QZ conceived and designed the experiment. XLZ, RBL, and RWJ performed the in vitro and in vivo experiments, Western blot and immunohistochemistry experiments. RBL and BFZ completed the Transwell invasion assay and H&E staining, and XLZ, RBL, and QZ performed the bioinformatics analysis. XLZ, RBL, and RWJ contributed to the analysis of the results and to the draft of the manuscript. XLZ, RWJ, and QZ wrote the manuscript. All authors read and approved the final manuscript.
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This animal study was reviewed and approved by the institutional ethical committee (Permit Number:SYXK 2009–0001). The HCC and adjacent tissue patient biopsies were approved by the Tianjin Medical University Cancer Hospital Ethics Committee.
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Zhou, X., Li, R., Jing, R. et al. Genome-wide CRISPR knockout screens identify ADAMTSL3 and PTEN genes as suppressors of HCC proliferation and metastasis, respectively. J Cancer Res Clin Oncol 146, 1509–1521 (2020). https://doi.org/10.1007/s00432-020-03207-9
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DOI: https://doi.org/10.1007/s00432-020-03207-9