Background/Aims: This study aims to investigate the effect of Luteolin on breast cancer in vitro and in vivo and the interaction between miRNAs and Notch signaling after Luteolin intervention, and illustrates the possible underlying mechanism and regulation loop. Methods: Cell growth/survival assays and cell cycle analyses were performed to evaluate cell survival in vitro. Scratch tests, cell invasion assays and tube formation assays were carried out to analyze cell viability and identify the impact of Luteolin on angiogenesis. Critical components in the Notch pathway including proteins and mRNAs were detected by Western blotting analyses, ELISA assays and real-time reverse transcription-polymerase chain reaction. Matrix metalloproteinases activity was evaluated by gelatin zymography analyses. MiRNAs were analyzed by miRNA expression assays. After MDA-MB-231 cells were separately transfected with Notch-1 siRNA/cDNA and miRNA mimics, the above assays were also carried out to examine potential tumor cell changes. Xenograft models were applied to evaluate the treatment potency of Luteolin in breast cancer. Results: Luteolin significantly inhibited breast cancer cell survival, cell cycle, tube formation and the expression of Notch signaling-related proteins and mRNAs, and regulated miRNAs. After introducing Notch-1 siRNA and miRNA mimics, MDA-MB-231 cells presented with changes in miRNA levels, reduced Notch signaling-related proteins, and decreased tumor survival, invasion and angiogenesis. Conclusion: Luteolin inhibits Notch signaling by regulating miRNAs. However, the effect of miRNAs on the Notch pathway could be either Luteolin-dependent or Luteolin-independent. Furthermore, Notch-1 alteration may inversely change miRNAs levels. Our data demonstrates that Luteolin, miRNAs and the Notch pathway are critical in breast cancer development and prognosis.

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Circular RNAs (circRNAs) are increasingly gaining importance and attention due to their diverse potential functions and their value as diagnostic biomarkers (disease specific). This study aims to explore the novel mechanisms by which exosome-contained circRNAs promote tumor development and metastasis in TNBC. We identified increased circRNA circPSMA1 in TNBC cells, their exosomes, and serum exosomes samples from TNBC patients. The overexpression of circPSMA1 promoted TNBC cell proliferation, migration, and metastasis both in vitro and in vivo. Moreover, we investigated the tumor-infiltrating immune cells (TICs) or stromal components in immune microenvironment (IME), and identified the significant differences in the immune cells between TNBC and non-TNBC samples. Mechanistically, circPSMA1 acted as a “miRNAs sponge” to absorb miR-637; miR-637 inhibited TNBC cell migration and metastasis by directly targeted Akt1, which recognized as a key immune-related gene and affected downstream genes β-catenin and cyclin D1. Subsequent co-culture experiments also demonstrated that exosomes from TNBC carrying large amounts of circPSMA1 could transmit migration and proliferation capacity to recipient cells. Kaplan–Meier plots showed that high expression of Akt1 and low expression of mir-637 are highly correlated with poor prognosis in patients with lymph node metastasis of TNBC. Collectively, all these results reveal that circPSMA1 functions as a tumor promoter through the circPSMA1/miR-637/Akt1-β-catenin (cyclin D1) regulatory axis, which can facilitate the tumorigenesis, metastasis, and immunosuppression of TNBC. Our research proposes a fresh perspective on novel potential biomarkers and immune treatment strategies for TNBC.