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To investigate the effects of Mucin 1 (MUC1) in human triple-negative breast cancer MDA-MB-231cells, the MDA-MB-231 cell line with MUC1 knockout (231-MUC1-KO) was constructed by CRISPR/Cas9 gene editing. Cell proliferation was evaluated using EDU and colony formation assays, and cell migration and invasion were detected by transwell assay. Autophagy flow was assessed by western blot and Ad-mCherry-GFP-LC3B dual-fluorescence system and validated by lysosome inhibitor barfimycin A1 and autophagy inducer rapamycin. Key proteins of autophagosomes and lysosomal fusion (STXl7, SNAP29) and lysosomal tagged protein (LAMP1) were detected by western blot, and lysosomal pH was evaluated by Lysotracker Red fluorescence. MUC1 expression was low in human normal breast epithelial cells MCF-10A, but was highly expressed in human MDA-MB-231 cells and tissues. Successful MUC1 knockout was confirmed by gene sequencing, RT-qPCR, and western blot. Loss of MUC1 gene expression in 231-MUC1-KO significantly reduced proliferation, migration, and invasion. Compared with the control group, MUC1 knockout led to a significant increase of autophagy-related proteins LC3-II and p62, which is consistent with the effect of lysosome inhibitor bleomycin A1. After adding the autophagy inducer rapamycin, compared with the control group, the accumulation of LC3-II and p62 proteins also further increased. The expression level of LAMP1 was downregulated and the lysosome pH increased, but the expression levels of STXl7 and SNAP29 were not affected. These findings suggest that MUC1 promotes malignant behaviors in MDA-MB-231 cells by regulating autophagic flow, likely through lysosomal dysfunction-mediated autophagy blockade.