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.

中文翻译：

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Withaferin A 介导乳腺癌衍生的乳腺球中 miRNA 表达的变化

乳腺癌是一种异质性疾病，由非典型细胞群组成，这些细胞群具有与治疗抗性、疾病复发和不良临床结果相关的干细胞样特征。微小 RNA (miRNA) 和小非编码 RNA 在调节自我更新、干性和细胞分化中起关键作用。Withaferin A (WA) 是一种甾体内酯，是Withania somnifera的主要生物活性成分（茄科）以其抗癌特性而闻名。在本研究中，利用小 RNA 测序评估了 WA 对乳腺癌衍生的乳腺球中 miRNA 表达的调节作用。用 WA 处理可抑制 MCF-7 和 T47D 细胞衍生的乳腺球形成，CD44、EpCAM、Nanog、OCT4 和 SOX2 作为自我更新和干细胞的标志物显着降低。小 RNA 测序显示在 WA 处理的 MCF-7 微球中总共有 395 个差异表达的 miRNA (DEM)，包括 194 个上调和 201 个下调的 miRNA。利用 KEGG 通路、基因本体富集、蛋白质-蛋白质和 miRNA-mRNA 相互作用网络的生物信息学分析确定了一些中枢基因的表达改变，即。 AKT1、PTEN、MYC、CCND1、  VEGFA、  NOTCH1和 IGFR1 与 WA 处理的乳腺球中的 DEM 相关。进一步的定量 RT-PCR 分析验证了 DEM 的表达，包括 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 和 let-7a-5p。特别是，let-7c-5p 被指定为乳腺癌中的肿瘤抑制因子。在 WA 处理后注意到 miR-let-7c-5p 表达增加，同时 CCND1 和 c-MYC 在 mRNA 和蛋白质水平上降低。总之，我们的研究证明了 WA 介导的 miRNA 表达，特别是 miR-let-7c-5p 的上调，导致抑制乳腺癌细胞衍生的乳腺球。