engleski

Generation of the GLI1, GLI2 and GLI3 knock-out ovarian cancer cell lines using the CRISPR/Cas9

INTRODUCTION Hedgehog signaling pathway is a developmental signaling pathway which is dormant in most adult differentiated tissues, but aberrantly activated in various tumors. In ovarian tumors it can be activated in a canonical way, by the SHH ligand, or the non-canonical way, by upregulation of the GLI transcription factors. Expression of GLI1 is usually associated with tumor progression in a clinical setting, but GLI2 and GLI3 also play a role by modifying the activity of GLI1 and transcription of their common transcriptional targets. In ovarian cancer, GLI3 protein is expressed in the full- length activator form, and not the shortened repressor form which is the predominant form for GLI3 protein. MATHERIAL AND METHOD To study the roles of the three GLI proteins in detail, we used the CRISPR/Cas9 guided gene editing to generate knock-out lines for each of the three GLI proteins. The sgRNA were designed using the online tool at crispr.mit.edu web site, and cloned into the pX330 vector expressing the sgRNA and the Cas9 protein. The sgRNA were designed to target the region close to the ATG site of each of the three GLI proteins, and after Cas9 nuclease activity the double stranded DNA break should be repaired by non-homologous end joining, which generates indels and leads to frameshift. Frameshift close to the ATG often leads to early termination of the protein, generating a knock- out. The SKOV3 ovarian cancer cell line was transfected with each of the vectors, the cells were split to the density of a single cell/well in a 96-well plate, and cell lines were expanded from single cells to obtain homogenous lines. RESULTS AND DISCUSSION SKOV3 ovarian cancer cell lines with knock outs in each of the three GLI proteins, GLI1, GLI2 and GLI3 were generated. The knock-out was confirmed by qRT-PCR and Western blot, and DNA was sequenced to show the exact genetic modification of the lines leading to the knock- out. The modified cell lines showed differences in cell morphology in relation to the original SKOV3 cell line immediately after expansion from the single cell. CONCLUSION The Hedgehog signaling pathway plays an important role in ovarian cancer. Cells harboring knock out for the effector proteins of the Hedgehog signaling pathway, GLI1, GLI2 and GLI3 show distinct changes in cell morphology and viability. The Hedgehog signaling pathway, and more specifically GLI proteins, may be a good potential therapeutic target in management of ovarian cancer.

CRISPR/Cas9, Hedgehog signaling, ovarian cancer

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