LAUSR

Breast cancer is a heterogeneous disease consisting of atypical cell populations that share stem cell‐like characteristics associated with therapeutic resistance, disease relapse, and poor clinical outcome. MicroRNAs (miRNA), and small noncoding RNA, are pivotal in the regulation of self‐renewal, stemness, and cellular differentiation. Withaferin A (WA), a steroidal lactone, is a major bioactive constituent of Withania somnifera (Solanaceae) known for its anticancer properties. In this study, the effect of WA on modulation of miRNA expression in breast cancer‐derived mammosphere was assessed utilizing small RNA sequencing. Treatment with WA inhibited MCF‐7 and T47D cells derived mammosphere formation with a significant decrease in CD44, EpCAM, Nanog, OCT4, and SOX2 as markers of self‐renewal and stemness. Small RNA sequencing demonstrated a total of 395 differentially expressed miRNAs (DEMs) including 194 upregulated and 201 downregulated miRNAs in WA‐treated MCF‐7 mammospheres. Bioinformatics analysis utilizing the KEGG pathway, Gene Ontology enrichment, protein−protein, and miRNA‐mRNA interaction network identified altered expression in a few hub genes viz. AKT1, PTEN, MYC, CCND1, VEGFA, NOTCH1, and IGFR1 associated with DEMs in WA‐treated mammospheres. Further quantitative RT‐PCR analysis validated the expression of DEMs including miR‐549a‐5p, miR‐1247‐5p, miR‐124‐5p, miR‐137‐5p, miR‐34a‐5p, miR‐146a‐5p, miR‐99a‐5p, miR‐181a‐5p, let‐7c‐5p, and let‐7a‐5p. In particular, let‐7c‐5p is designated as a tumor suppressor in breast cancer. An increase in miR‐let‐7c‐5p expression was noted after WA treatment, with a simultaneous decrease in CCND1 and c‐MYC at mRNA and protein levels. Taken together, our study demonstrated WA‐mediated miRNA expression, in particular, upregulation of miR‐let‐7c‐5p, leads to the inhibition of breast cancer cells derived mammospheres.