Genomic deletion of linc۰۱۱۱۶ promoter using CRISPR/Cas۹ technique and determination of the linc۰۱۱۱۶knock-out effect on the breas t cancer cell proliferation

Objective: Linc۰۱۱۱۶ has been shown to increase breas t cancercell proliferation, the severity of malignancy, invasion, and metastasis. Therefore, this LncRNA is being proposed that playsan oncogenic role and also is sugges ted as a prognos tic andtherapeutic target in breas t cancers. The present s tudy aimedto specifically target the linc۰۱۱۱۶ promoter sequence in breas tcancer cell lines using the CRISPR/Cas۹ technique. In the following,the efficiency of this method in the targeted deletion ofgenomic sequence and thus the inhibition of mentioned LncRNAtranscription activity will be examined.Materials and Methods: Firs t, the expression level of linc۰۱۱۱۶in the non-malignant breas t epithelial cells (MCF۱۰A), as wellas tumor cells (MDM MB۲۳۱, MCF۷, and SKBR۳), were evaluatedby RT-qPCR. Then, two separate plasmids with mCherryand EGFP reporters were cons tructed harboring sgRNA codingsequences and Cas۹ protein which were subsequently co-transfectedby electroporation into the target cells. Finally, transfectedcell lines were examined to determine genomic changes, theexpression level of linc۰۱۱۱۶, cell proliferation, and invasion rate.Results: The results demons trated that the Linc۰۱۱۱۶ expressionlevel was significantly increased in breas t cancer linescompared with normal breas t cells. The accuracy of cons tructedvectors was confirmed using PCR, sequence analysis, and restriction diges tion. Flow cytometry and fluorescent microscopedetermined the expression of the EGFP and mCherry reportergenes. Also, the efficient deletion of target sequences was revealedby genomic PCR.Conclusion: Based on the results, growth rate, proliferation,and invasion of breas t cancer cells were reduced as a consequenceof Linc۰۱۱۱۶ knockout through the CRISPR/Cas۹ system.Thus, targeting this oncogenic lncRNA has a promisingadvantage in the effective diagnosis and treatment of breas tcancer.

کلیدواژه ها:

Breas t Cancer ، CRISPR/Cas۹ ، Linc۰۱۱۱۶