Kruppel like factor 4 (KLF4), a transcription factor associated with carcinogenesis and tumor progression, plays an important role in various malignancies. In the present study, we utilized the CRISPR-ON system to upregulate KLF4 expression level and subsequently investigated the effect and mechanism of KLF4 in the carcinogenesis and progression of urothelial bladder cancer (UBC). Immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) were used to evaluate the expression of KLF4. The CpG methylation status of the promoter region was analyzed using bisulfite-sequencing PCR (BSP). CRISPR-ON system comprised sgRNA and dCas9 protein combined with a transcriptional activation domain. The cell proliferation and cell cycle were assessed by CCK-8 assay, flow cytometry and colony formation assay. The cell motility ability was evaluated using trans-well assay. In vivo tumorigenesis assay and lung metastasis model were also performed. The KLF4 expression was significantly downregulated in UBC tissues. The high CpG methylation status in the promoter of KLF4 was confirmed using BSP. KLF4 overexpression was successfully achieved via CRISPR-ON system, which inhibited the proliferation and induced G1-phase arrest in T24 cells through the regulation of AKT/p21 signal. Furthermore, enforced expression of KLF4 also abrogated the migration and invasion of T24 cells by suppressing EMT progression. Finally, in vivo models indicated that the upregulation of KLF4 could inhibit tumorigenesis and lung metastasis in nude mice. In conclusion, KLF4 overexpression mediated by CRISPR-ON inhibits tumorigenesis and EMT progression in UBC cells, representing a potential therapeutic target, and CRISPR-ON system could be a therapeutic strategy for UBC in the future.
Oncotarget. 2017 Oct 27*** epublish ***
Xin Xu, Jiangfeng Li, Yi Zhu, Bo Xie, Xiao Wang, Song Wang, Haiyun Xie, Huaqing Yan, Yufan Ying, Yiwei Lin, Ben Liu, Wei Wang, Xiangyi Zheng
Department of Urology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, P.R. China., Department of Urology, Tongde Hospital of Zhejiang Province, Hangzhou 310012, Zhejiang Province, P.R. China.