CRISPR-ON-Mediated KLF4 overexpression inhibits the proliferation, migration and invasion of urothelial bladder cancer in vitro and in vivo

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.

Keywords: CRISPR-ON; EMT; KLF4; proliferation; urothelial bladder cancer.

Figure 1. KLF4 is downregulated in UBC tissues and cells

(A) The results of q-RT-PCR revealed a lower relative KLF4 expression in UBC cells (UM-UC-3 and T24) compared with a normal bladder cell line (SV-HUC-1). (B) Typical images of IHC of TMA. KLF4 was localized in the cytoplasm. (C) Statistical analysis showed the expression level of KLF4 protein in UBC tissues was aberrantly lower than in adjacent non-tumor tissues. (D) Kaplan–Meier survival analysis indicated that the higher protein expression of KLF4 was significantly associated with a higher OS rate in UBC patients. The data were expressed as the means ± S.D. ^*P < 0.05.

Figure 2. The CRISPR-ON system upregulated the expression level of KLF4

(A) The constructed lentiviral plasmid diagrams were shown: lenti-Dcas9-vp64-Puro and lenti-sgRNA-MS2-P65-HSF1-Neo. (B) Three sgRNA sequences were designed. (C) The results of qRT-PCR suggested that the relative mRNA expression level of KLF4 was obviously upregulated by CRISPR-ON system. (D) The Western blot analysis showed the upregulation of KLF4 protein expression by CRISPR-ON system. The data were expressed as the means ± S.D. ^* P < 0.05.

Figure 3. Overexpressed KLF4 abrogated the proliferation and induced G1-phase arrest in T24 cells

(A) The cell growth curve showed the retard of T24 cells treated with SAM fused with sgRNA-KLF4(3) compared with NC. (B) The colony formation assay also indicated the reduced colony rate in T24 cells treated with SAM fused with sgRNA-KLF4(3). (C) The colony count was calculated. (D) The cell cycle assay revealed G1-phase arrest in T24 cells treated with SAM fused with sgRNA-KLF4(3). (E) The percentages of cells in different phases were calculated. (F) Overexpressed KLF4 inhibited the proteins associated with the AKT/p21 signal. The data were expressed as the means ± S.D. ^* P < 0.05.

Figure 4. Overexpressed KLF4 inhibited invasion and migration in T24 cells

(A) The results of trans-well assay (representative micrographs are presented) demonstrated the inhibition of invasion and migration in the sgRNA-KLF4(3) group. (B) The relative invasion and migration rate was calculated. (C) A wound healing assay indicated that the overexpression of KLF4 in T24 cells resulted in a retardation of wound closure compared with the control group. (D) EMT progression was suppressed in the sgRNA-KLF4(3) group. The data were expressed as the means ± S.D. ^* P < 0.05.

Figure 5. KLF4 inhibited tumor growth and lung metastasis in vivo

(A) The growth of tumor xenografts originated from T24-KLF4(3) cells was retarded compared with that in the NC group. (B) The tumor xenograft was dissected from the sacrificed naked mice. (C) The tumor weight was measured and compared. (D) H&E staining of mouse lungs. Lung metastatic foci was indicated with a black arrow. The data were expressed as the means ± S.D. ^* P < 0.05.

Figure 6. High CpG methylation status in the promoter of KLF4 was confirmed using BSP

(A) The regions analyzed using BSP were indicated. (B) qRT-PCR indicated that 5-aza-dc treatment promoted the expression of KLF4 in both T24 and UM-UC3 cells. (C) Tumor tissues and cell lines (T24 and UM-UC-3) possessed higher methylation sites than normal bladder tissues. (D) The methylated CpG rate was calculated and compared. The data were expressed as the means ± S.D. ^* P < 0.05.