miR-99b-5p inhibition drives apoptosis and tumor shrinkage in triple-negative breast cancer: functional characterization through AGO2-RIP-seq and mechanistic insights.

Background: Dysregulated microRNAs (miRNAs) are critical contributors to breast cancer biology, yet the functional roles of many remain incompletely understood. miR-99b-5p has been widely characterized as a tumor-suppressive miRNA in numerous cancer types, where its expression is consistently reduced in tumors compared with normal tissues. In contrast, our analyses of breast cancer datasets revealed a unique expression pattern: miR-99b-5p is significantly upregulated in breast tumors, suggesting a context-dependent oncogenic function. In this study, we identified miR-99b-5p as an oncogenic driver in triple-negative breast cancer (TNBC). Methods and results: TCGA-based expression profiling confirmed its elevated levels in breast tumors. Functional assays demonstrated that downregulation of miR-99b-5p in TNBC cells inhibits proliferation and induces apoptosis, indicating a critical role in sustaining tumor cell survival. To elucidate the molecular mechanisms underlying this activity, we performed AGO2-RNA immunoprecipitation followed by high-throughput sequencing (AGO2-RIP-Seq), enabling unbiased identification of miR-99b-5p-associated transcripts. Pathway enrichment analyses revealed that its direct targets converge on apoptotic regulation, cell-cycle control, and ubiquitin-mediated protein degradation. Mechanistic validation through qRT-PCR, Western blotting, and luciferase assays confirmed that miR-99b-5p modulates the TRAIL-R signaling pathway via DR5 and BAK, attenuating apoptotic signaling. Conclusion: Collectively, these findings establish miR-99b-5p as a context-specific oncogenic miRNA in breast cancer and a promising therapeutic target, particularly for TNBC, where targeted treatment options remain limited.