目的:鉴定和验证功能上解释前列腺癌生长和转移的基因。
方法:通过用全基因组敲除文库包装的慢病毒转染DU145细胞来构建DU145-KO细胞系。将DU145-KO细胞移植到免疫受损的Nu/Nu小鼠的腋下,然后在第3周(早期肺组织)或第7周(具有微转移的晚期肺组织)从肺收集组织,以及接种后第7周(晚期原发肿瘤)的原发肿瘤部位。在不同的时间点检索肺转移进行DNA测序分析,以鉴定富集的sgRNA,因此候选基因/miRNA。进行了进一步的生物信息学分析和有限的功能验证研究。
结果:DU145-KO细胞促进小鼠移植瘤的形成,促进原发肿瘤的生长和转移,与对照(DU145-NC细胞)相比。序列数据分析显示,在原发肿瘤和微转移部位,sgRNA的丰度发生了显著变化。15个靶基因(C1QTNF9B,FAM229A,hsa-mir-3929,KRT23,TARS2,CRADD,GRIK4,PLA2G15,LOXL1,SLITRK6,CDC42EP5,SLC2A4,PTGDS,MYL9和ACOX2用于富集的sgRNA)已被选择用于实验验证,这表明任何这些基因的敲除导致DU145细胞的侵袭和转移潜力增强。
结论:全基因组CRISPR-Cas9敲除筛选技术结合高通量测序分析鉴定出可能与前列腺肿瘤侵袭和转移相关的基因。对这些基因的分析提供了对与疾病相关的生物学途径的见解,并公开了用于诊断或预后的创新标志物以及潜在的治疗靶标。
Identification and validation of genes that functionally account for the growth and metastasis of prostate cancer.
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.
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.
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.