Abstract
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.
Highlights
Urothelial bladder cancer (UBC) is the ninth most frequently-diagnosed cancer worldwide, with an estimated 430,000 new cases diagnosed in 2012 and a mortality rate ranking 13th in terms of deaths [1, 2]
Kruppel like factor 4 (KLF4) overexpression mediated by clustered regularly interspaced short palindromic repeats (CRISPR)-ON inhibits tumorigenesis and EMT progression in urothelial bladder cancer (UBC) cells, representing a potential therapeutic target, and CRISPRON system could be a therapeutic strategy for UBC in the future
Kaplan-Meier survival curves indicated that the KLF4 expression was closely associated with the overall survival (OS) rate in UBC patients (Figure 1D)
Summary
Urothelial bladder cancer (UBC) is the ninth most frequently-diagnosed cancer worldwide, with an estimated 430,000 new cases diagnosed in 2012 and a mortality rate ranking 13th in terms of deaths [1, 2]. As illustrated by Barrangou R et al, the specific effect and mechanism of CRISPR was to provide resistance against exogenous virus together with associated CAS genes [5]. The CRISPR/ CAS9 system was gradually engineered and utilized as a gene-editing tool in human cells [6, 7]. Jaenisch et al created a CRISPR-ON system comprising a nucleasedead Cas (dCas9) protein combined with a transcriptional activation domain and single guide RNAs (sgRNAs) with sequences complementary to the gene promoter. They have demonstrated that CRISPR-ON can efficiently upregulate exogenous reporter genes in both human and mouse cells [8]. CRISPR-ON has the characteristics of robustness and specificity, and can facilitate genome scale gain-of-function (GOF) screening [8, 9]
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