miR-99b-5p as a Modulator of KLF4 Expression and Drug Response in Luminal B Breast Cancer via a Ubiquitin-Mediated Mechanism

Ubiquitin-Mediated KLF4 Regulation by miR-99b-5p

Abstract

Objective: Luminal B breast cancer (BC) exhibits aggressive behavior and distinct molecular features, often leading to resistance to therapies such as trastuzumab and tamoxifen. MicroRNAs have emerged as key regulators of cancer progression and drug response. This study explores the role of miR-99b-5p in modulating treatment resistance in Luminal B BC.

Methods: Both trastuzumab-sensitive BT-474 cells and trastuzumab-resistant BT-474 cells were employed to evaluate the functional role of miR-99b-5p through mimic and inhibitor transfections. Gene and protein expression levels were measured using qRT-PCR and Western blotting, respectively. Bioinformatic analyses were performed using the ENCORI, UALCAN, GeneMiner, ROCplot, and UbiBrowser databases.

Results: Our results demonstrated that miR-99b-5p expression was initially downregulated following tamoxifen or trastuzumab treatment, but was subsequently elevated in trastuzumab-resistant cells, suggesting a dynamic regulatory role in therapeutic adaptation. Bioinformatic analyses identified an association between miR-99b-5p and Kruppel-like factor 4 (KLF4), a zinc-finger transcription factor implicated in BC progression and therapy sensitivity. Biological assays provided evidence that miR-99b-5p positively regulates KLF4 and BCL2 protein levels, potentially promoting cell survival and contributing to drug resistance. Mechanistically, we identified TRAF7 as a downstream target of miR-99b-5p, and TRAF7 modulates KLF4 protein stability through ubiquitin-mediated degradation. Suppression of TRAF7 by miR-99b-5p reduces KLF4 ubiquitination, enhancing its stability and downstream signaling.

Conclusion: These findings reveal a novel miR-99b-5p/TRAF7/KLF4 axis that promotes drug resistance in Luminal B BC through post-transcriptional and ubiquitin-mediated mechanisms. Targeting this regulatory pathway may represent a promising strategy to overcome therapy resistance and may warrant further investigation in biomarker development and targeted treatment design.