Zhen Shuai, Hua Ling, Liu Yun-Hui, Sun Xiao-Min, Jiang Meng-Meng, Chen Wei, Zhao Le, Li Xu
Center for Translational Medicine, The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710061, P.R. China.
Key Laboratory for Tumor Precision Medicine of Shaanxi Province, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, P.R. China.
Oncotarget. 2017 Feb 7;8(6):9634-9646. doi: 10.18632/oncotarget.14176.
CRISPR/Cas9 is a novel and effective genome editing technique, but its application is not widely expanded to manipulate long non-coding RNA (lncRNA) expression. The lncRNA urothelial carcinoma-associated 1 (UCA1) is upregulated in bladder cancer and promotes the progression of bladder cancer. Here, we design gRNAs specific to UCA1 and construct CRISPR/Cas9 systems targeting UCA1. Single CRISPR/Cas9-UCA1 can effectively inhibit UCA1 expression when transfected into 5637 and T24 bladder cancer cells, while the combined transfection of the two most effective CRISPR/Cas9-UCA1s can generate more satisfied inhibitory effect. CRISPR/Cas9-UCA1s attenuate UCA1 expression via targeted genome-specific DNA cleavage, resulting in the significant inhibition of cell proliferation, migration and invasion in vitro and in vivo. The mechanisms associated with the inhibitory effect of CRISPR/Cas9-UCA1 on malignant phenotypes of bladder cancer are attributed to the induction of cell cycle arrest at G1 phase, a substantial increase of apoptosis, and an enhanced activity of MMPs. Additionally, urinary UCA1 can be used as a non-invasive diagnostic marker for bladder cancer as revealed by a meta-analysis. Collectively, our data suggest that CRISPR/Cas9 technique can be used to down-modulate lncRNA expression, and urinary UCA1 may be used as a non-invasive marker for diagnosis of bladder cancer.
CRISPR/Cas9是一种新颖且有效的基因组编辑技术，但其应用尚未广泛扩展至操纵长链非编码RNA（lncRNA）的表达。lncRNA尿路上皮癌相关1（UCA1）在膀胱癌中上调，并促进膀胱癌的进展。在此，我们设计了针对UCA1的gRNA，并构建了靶向UCA1的CRISPR/Cas9系统。当将单个CRISPR/Cas9-UCA1转染到5637和T24膀胱癌细胞中时，它可以有效抑制UCA1的表达，而联合转染两个最有效的CRISPR/Cas9-UCA1可以产生更满意的抑制效果。CRISPR/Cas9-UCA1通过靶向基因组特异性DNA切割来减弱UCA1的表达，从而在体外和体内显著抑制细胞增殖、迁移和侵袭。CRISPR/Cas9-UCA1对膀胱癌恶性表型的抑制作用相关机制归因于诱导细胞周期停滞在G1期、凋亡的大量增加以及基质金属蛋白酶活性的增强。此外，一项荟萃分析显示，尿液中的UCA1可作为膀胱癌的非侵入性诊断标志物。总体而言，我们的数据表明CRISPR/Cas9技术可用于下调lncRNA的表达，并且尿液UCA1可能用作膀胱癌诊断的非侵入性标志物。
