Withaferin A mediated changes of miRNA expression in breast cancer‐derived mammospheres

Abstract

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.

Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells

“…RNA concentration and purity were estimated using a Nanodrop200 spectrophotometer (Thermo Fisher Scientific, USA) and further validated by a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA). The RNA integrity was determined using a 2100 Bioanalyzer (Agilent 2100 expert) and checked by an Agilent 2100 Bioanalyzer using a RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA) [ 21 ].…”

“…The miRNAs with log2 fold > 1.5 were considered as “UP” regulated; miRNAs with log2 fold < −1.5 were considered as “DOWN” regulated. The volcano plots for differentially expressed miRNAs (DEMs) were generated using GraphPad prism software 5 [ 21 ].…”

“…The predicted target genes were submitted to the online Database for Annotation, Visualization, and Integrated Discovery (DAVID) program for Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis. Only the significant ( p < 0.05) GO term and KEGG pathways were considered [ 21 ].…”

Naphthylisoindolinone alkaloids: the first ring-contracted naphthylisoquinolines, from the tropical liana Ancistrocladus abbreviatus, with cytotoxic activity

“…52–54 These cancer-stem-like cells have been identified in a variety of tumors and are under suspicion to be a causative factor of tumor growth and metastasis, eventually facilitating resistance to chemotherapies, and thus, leading to treatment failure and tumor recurrence. 55 Therefore, targeting these cancer stem cells in breast cancer has become a promising approach. 56…”

“…[52][53][54] These cancer-stem-like cells have been identied in a variety of tumors and are under suspicion to be a causative factor of tumor growth and metastasis, eventually facilitating resistance to chemotherapies, and thus, leading to treatment failure and tumor recurrence. 55 Therefore, targeting these cancer stem cells in breast cancer has become a promising approach. 56 Cultivation of human breast epithelial adenocarcinoma cells MCF-7 under non-adherent non-differentiating conditions as described in the Experimental section results in the generation of discrete clusters of spheroids, termed mammospheres (Fig.…”