Objective: Identification and validation of genes that functionally account for the growth and metastasis of prostate cancer.
Methods: DU145-KO cell line was constructed by transfecting DU145 cells with lentivirus packaged with the genome-wide knock-out library. The DU145-KO cells were transplanted into the armpits of immunocompromised Nu/Nu mice, followed by the tissue collection from the lung at week 3 (early lung tissue) or week 7 (late lung tissue with micro-metastasis), as well as from primary tumor site at week 7 (late primary tumor) after inoculation. Lung metastasis was retrieved at various time points for DNA sequencing analysis to identify enriched sgRNAs, thus candidate genes/miRNAs. Further bioinformatics analysis and limited functional validation studies were carried out.
Results: DU145-KO cells promoted the formation of transplanted tumors in mice and promoted the growth and metastasis of primary tumors, compared to the controls (DU145-NC cells). The analysis of sequence data showed that the abundance of sgRNAs significantly changed in the primary tumor and micro-metastasis site. Fifteen target genes (C1QTNF9B, FAM229A, hsa-mir-3929, KRT23, TARS2, CRADD, GRIK4, PLA2G15, LOXL1, SLITRK6, CDC42EP5, SLC2A4, PTGDS, MYL9 and ACOX2 for the enriched sgRNAs) have been selected for experimental validation, which showed that knock-out of any of these genes led to the enhanced potential of invasion and metastasis of DU145 cells.
Conclusion: Genome-wide CRISPR-Cas9 knock-out screening technology combined with highthroughput sequencing analysis identified genes that potentially relate to prostate tumor invasion and metastasis. Analysis of these genes provided insights into biological pathways relevant to the disease and disclosed innovative markers for diagnosis or prognosis as well as potential targets for therapy.
[http://dx.doi.org/10.1128/jb.169.12.5429-5433.1987] [PMID: 3316184]
[http://dx.doi.org/10.1111/j.1365-2958.1993.tb01721.x] [PMID: 8412707]
[http://dx.doi.org/10.1073/pnas.0607117103] [PMID: 17030793]
[http://dx.doi.org/10.1186/1745-6150-1-7] [PMID: 16545108]
[http://dx.doi.org/10.1126/science.1159689] [PMID: 18703739]
[http://dx.doi.org/10.1016/j.celrep.2015.06.049] [PMID: 26190106]
[http://dx.doi.org/10.1016/j.cell.2015.06.059] [PMID: 26189680]
[http://dx.doi.org/10.1016/j.febslet.2006.03.041] [PMID: 16580667]
[http://dx.doi.org/10.1002/pros.20483] [PMID: 16835890]
[http://dx.doi.org/10.1074/jbc.RX120.015588] [PMID: 32917829]
[http://dx.doi.org/10.1016/B978-0-12-418687-3.00018-5] [PMID: 24011049]
[http://dx.doi.org/10.1038/laban.254] [PMID: 23689461]
[http://dx.doi.org/10.1155/2014/230183] [PMID: 25197632]
[http://dx.doi.org/10.1186/s13059-017-1237-8] [PMID: 28615073]
[http://dx.doi.org/10.1038/cr.2017.81] [PMID: 28585534]
[http://dx.doi.org/10.1146/annurev-biophys-062215-010822] [PMID: 28375731]
[http://dx.doi.org/10.1016/j.cell.2015.02.038] [PMID: 25748654]
[http://dx.doi.org/10.1038/s41422-018-0020-z] [PMID: 29507396]
[http://dx.doi.org/10.1016/j.pharmthera.2017.08.009] [PMID: 28834698]