The effect of the deletion of MIEN1 promoter region in colorectal cancer cell line HT29.

Purpose: MIEN1 (Migration and Invasion Enhancer 1 and MIEN1), located in chromosome 17q12, encodes a membraneandanchored protein that functions as an oncoprotein in several types of cancers (breast, prostate, oral and colorectal cancer among others), enhancing the biological processes of migration and invasion. The aim of this project is to evaluate the effect of the deletion of the promoter region of MIEN1 on migration, invasion and cell adhesion. Methods :The promoter of MIEN1 was deleted in the colorectal cancer cell line HT29, through CRISPR-Cas9. The region deleted spanned 856bp (-870pb to -15pb). Sanger sequencing, qPCR and western blot confirmed the deletion of the promoter region and the knockout of the gene, respectively. The expression of HER2 and GRB7 (ERBB2 amplicon) was evaluated by western blot. Migration by soft agar beads assay, invasion by Boyden chamber and cellular adhesion by hanging drop assay were performed to determine the effect on the knockout cells. Results: The genome editing approach only abolished the expression of MIEN1, since the expression of the surrounding genes was not affected. HT29 knockout cells showed a decrease in migration (44%) and invasion capacity (43%) as compared to HT29 wild type. Also, in the cell adhesion assay, a decrease in the size of cell aggregates was observed in the knockout cell line (30%). Conclusions: The ablation of MIEN1 promoter in HT29 cells affected several cellular processes related to metastasis. Further analysis of the molecular mechanisms will be presented.