Proliferation and Invasion Inhibition of ColorectalCancer Cells by Knocking Out The MSI۱ Gene UsingCRISPR/Cas۹ Technique

Objective: Colorectal cancer is one the mos t common cancersworldwide. The RNA-binding protein Musashi۱ (MSI۱) playsan important role in the regulating of the cell differentiation incolon progenitor cells. MSI۱ overexpression has been reportedin several malignances including colorectal cancer. Overexpressionof the MSI۱ gene increases cell division and invasion,but decreased expression of MSI۱ leads to reduced cell proliferationand invasion in a variety of malignancies. The aim ofthis s tudy was to knock out the MSI۱ gene in colorectal cancercells by the CRISPR/Cas۹ technique and seek its effect on proliferationand invasion rate of the cancer cells.Materials and Methods: Firs t, appropriate sgRNAs targetingMSI۱ gene was designed bioinformatically. HCT۱۱۶ cells withthe highes t expression of MSI۱ were used for knocking out.Two vectors, one containing the specific sgRNAs sequences,and the other with Cas۹ coding sequence were cons tructed andco-transfected into the HCT۱۱۶ cells by lipofection. The functionof Cas۹ in specific deletion of the MSI۱ genomic region was determined using polymerase chain reaction (PCR) andsequencing. Finally, the effect of decreasing MSI۱ expressionon proliferation and invasion of the colorectal cancer cells wereinves tigated.Results: The accuracy of cons tructed vectors was confirmedusing PCR and sequence analysis. Flow cytometry and fluorescentmicroscope also showed the successful co-transfection ofHCT۱۱۶ cells due to expression of EGFP and mCherry reporters.The efficient deletion of target sequences was also verifiedusing genomic PCR. Finally, the results showed that colorectalcancer cell proliferation and invasion were significantly reducedcompared to untreated cells as the control.Conclusion: Based on our findings, targeting and disruption ofMSI۱ as an oncogene could be a promising approach to improvethe cytotoxic effect agains t the colorectal cancer cells

کلیدواژه ها:

Colorectal Cancer ، Crispr/Cas۹ ، Knockout ، MSI۱ Gene