Knocking out the MSI۱ gene by CRISPR/Cas۹ technique reduced the proliferation and invasion ofthe colorectal cancer cells

Objective: Colorectal cancer is one the most common cancersworldwide. It has been shown that RNA-binding proteinMusashi۱ (MSI۱) plays an important role in regulating of colonprogenitor cells. Pathological overexpression of MSI۱ has beenreported in several malignances including colorectal cancer.The aims of the present study is to specific knockout of MSI۱gene in colorectal cancer cells by CRISPR/Cas۹ technique andobserve the effects of this manipulation on proliferation, survivaland invasion rate of the cancerous cells.Method: At first, two appropriate sgRNAs for targeting theMSI۱ gene was designed by several computational design toolssuch as www.crispor.tefor.net and www.benchling.com. Twopairs of sgRNAs, which were presented with the highest scoresby the mentioned tools, were selected for experimental analysis.Each pair of sgRNAs was subcloned into the px۴۵۹ vectorthat contained puromycin resistance and Cas۹ expressioncassette. In order to knock out the MSI۱ gene, px۴۵۹ vectorwas transfected into the HCT۱۱۶ cells by Lipofectamine LTX.Finally, the effects of decreasing MSI۱ expression on proliferationand invasion of the colorectal cancer cells were evaluatedin selected clones.Result: The accuracy of constructed vectors was confirmed usingPCR and sequence analysis. Genomic PCR and sequencingalso verified the efficient deletion of MSI۱ target sequences inthe manipulated HCT۱۱۶ cells. Finally, the results showed thatproliferation and invasion of colorectal cancer cells were significantlyreduced in the selected clones compared to the untreated cells as the negative control.Conclusion: This study indicated that targeting and disruptionof MSI۱ as an oncogene significantly suppressed the cancerousproperties of HCT۱۱۶ cells including proliferation, survival andinvasiveness. Consequently, MSI۱ gene can be considered as apromising molecular target such that shutting down of the genemay be a new approach to enhance the clinical outcomes fortreatment of colorectal cancer.

کلیدواژه ها:

Colorectal cancer ، HCT۱۱۶ ، MSI۱ gene ، CRISPR/Cas۹ ، Knockout