Cas9 Mediated Correction of β-catenin Mutation and Restoring the Expression of Protein Phosphorylation in Colon Cancer HCT-116 Cells Decrease Cell Proliferation in vitro and Hamper Tumor Growth in Mice in vivo.

Abstract

PurposeColorectal cancer (CRC) is one of the major contributors to cancer mortality and morbidity. Finding strategies to fight against CRC is urgently required. Mutations in driver genes of APC or β-catenin play an important role in the occurrence and progression of CRC. In the present study, we jointly apply CRISPR/Cas9-sgRNA system and Single-stranded oligodeoxynucleotide (ssODN) as templates to correct a heterozygous ΔTCT deletion mutation of β-catenin present in a colon cancer cell line HCT-116. This method provides a potential strategy in gene therapy for cancer.MethodsA Cas9/β-catenin-sgRNA-eGFP co-expression vector was constructed and co-transfected with ssODN into HCT-116 cells. Mutation-corrected single-cell clones were sorted by FACS and judged by TA cloning and DNA sequencing. Effects of CRISPR/Cas9-mediated correction were tested by real-time quantitative PCR, Western blotting, CCK8, EDU dyeing and cell-plated clones. Moreover, the growth of cell clones derived tumors was analyzed at nude mice xenografts.ResultsCRISPR/Cas9-mediated β-catenin mutation correction resulted in the presence of TCT sequence and the re-expression of phosphorylation β-catenin at Ser45, which restored the normal function of phosphorylation β-catenin including reduction of the transportation of nuclear β-catenin and the expression of downstream c-myc, survivin. Significantly reduced cell growth was observed in β-catenin mutation-corrected cells. Mice xenografted with mutation-corrected HCT-116 cells showed significantly smaller tumor size than uncorrected xenografts.ConclusionThe data of this study documented that correction of the driven mutation by the combination of CRISPR/Cas9 and ssODN could greatly remedy the biological behavior of the cancer cell line, suggesting a potential application of this strategy in gene therapy of cancer.