Objective:Anoikis is apoptosis that is induced when cells detach from the extracellular matrix and neighboring cells.As anoikis serves as a regulatory barrier,cancer cells often acquire resistance towards anoikis during tumorigenesis to become metastatic.MicroRNAs (miRNAs) are short strand RNA molecules that regulate genes post-transcriptionally by binding to mRNAs and reducing the expression of its target genes.This study aimed to elucidate the role of a novel miRNA,miR-6744-5p,in regulating anoikis in breast cancer and identify its target gene.Methods:An anoikis resistant variant of the luminal A type breast cancer MCF-7 cell line (MCF-7-AR) was generated by selecting and amplifying surviving cells after repeated exposure to growth in suspension.MiRNA microarray analysis identified a list of dysregulated miRNAs from which miR-6744-5p was chosen for overexpression and knockdown studies in MCF-7.Additionally,the miRNA was also overexpressed in a triple-negative breast cancer cell line,MDA-MB-231,to evaluate its ability to impair the metastatic potential of breast cancer cells.Results:This study showed that overexpression and knockdown of miR-6744-5p in MCF-7 increased and decreased anoikis sensitivity,respectively.Similarly,overexpression of miR-6744-5p in MDA-MB-231 increased anoikis and also decreased tumor cell invasion in vitro and in vivo.Furthermore,NAT1 enzyme was identified and validated as the direct target of miR-6744-5p.Conclusions:This study has proven the ability of miR-6744-5p to increase anoikis sensitivity in both luminal A and triple negative breast cancer cell lines,highlighting its therapeutic potential in treating breast cancer.