Dose-Dependent Downregulation of miR-335-5p in Response to Tamoxifen Reveals a PIAS4–ESR1 Regulatory Axis in Breast Cancer Cells

Breast cancer remains the most frequently diagnosed cancer among women worldwide, and estrogen receptor–positive (ER+) tumors are primarily treated with endocrine therapies; however, resistance to agents such as tamoxifen limits treatment efficacy. MicroRNAs (miRNAs) have emerged as pivotal regulatory molecules in breast cancer, offering promising avenues for both therapeutic intervention and prognostic evaluation. In this study, we investigated the role of miR-335-5p in response to tamoxifen and trastuzumab and explored its regulatory relationship with ESR1, NRIP1, and PIAS4. ER-positive (ER+) breast cancer cells were transfected with miR-335-5p mimic or scrambled control and treated with tamoxifen at IC50 and IC70 concentrations. Gene and miRNA expression levels were assessed using qRT-PCR, and protein expression was evaluated by Western blotting. Bioinformatics analyses, including target prediction, correlation analyses, and drug response datasets, were integrated to support the findings. Our results indicate that miR-335-5p regulates ESR1 and NRIP1 expression indirectly through PIAS4, a negative regulator of these genes. This interaction is consistent with the observed positive correlation between miR-335-5p and its downstream targets. Collectively, these findings suggest a dose-dependent role of miR-335-5p in modulating endocrine therapy response and identify a previously uncharacterized miR-335–ESR1–PIAS4 regulatory axis in ER+ breast cancer.