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

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.

In vivo studies using xenograft models established with MDA-MB-231 cells stably expressing miR-99b-5p knockdown showed marked tumor regression, further supporting its oncogenic role. 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.