EACR25-0160

miR-7974: A predictor of poor prognosis and link to autophagy in ER+ breast cancer

S. Eneh1, J. Hartikainen1,2,3, S. Heikkinen4, R. Sironen1,2,5, M. Tengström6, V. Kosma1,2,7, S. Ahuja1, A. Mannermaa1,2,7
1University of Eastern Finland, Institute of Clinical Medicine, Clinical Pathology and Forensic Medicine, School of Medicine, Kuopio, Finland
2University of Eastern Finland, Multidisciplinary Cancer Research Community (Cancer RC), Kuopio, Finland
3University of Eastern Finland, Institute of Clinical Medicine, Genome Center of Eastern Finland, School of Medicine, Kuopio, Finland
4University of Eastern Finland, Institute of Biomedicine, School of Medicine, Kuopio, Finland
5Kuopio University Hospital, Department of Clinical Pathology, Kuopio, Finland
6Kuopio University Hospital, Cancer Center, Department of Oncology, Kuopio, Finland
7Kuopio University Hospital, Biobank of Eastern Finland, Kuopio, Finland
Introduction:

Patients with estrogen receptor-positive (ER+) breast cancer (BC) generally have better survival rates than those with ER-negative early BC; however, individual prognoses can vary greatly. Therefore, identifying biomarkers to detect patients who may require more aggressive treatment is crucial. MicroRNAs (miRNAs), a class of small non-coding RNAs, regulate gene expression and have been linked to cancer progression, making them promising candidates for diagnosis and prognosis. In this study, we investigate miR-7974, a less-explored miRNA, and its potential role in ER+ BC.

Material and method:

We extracted total RNA from 204 breast tissue samples from the Kuopio Breast Cancer Study cohort using the mirVana™ miRNA Isolation Kit. We prepared small RNA sequencing libraries with the TruSeq® Small RNA Library Prep Kit and sequenced them on an Illumina MiSeq sequencer. To investigate the correlation between miR-7974 and clinical features, as well as patient outcomes in breast cancer (BC), we used bioinformatics tools like DESeq2 and multivariate survival analyses. To study the functional role of miR-7974 in MCF-7 at the molecular and cellular levels, we utilized in vitro methods, including Western blot, qPCR, and miRNA pull-down assays, alongside in ovo chick chorioallantoic membrane (CAM) assay. Additionally, the direct target of miR-7974 was validated using another BC cell line, MDA-MB-453.

Result and discussion:

Our findings show that the upregulation of miR-7974 was significantly (P=8.93x10^-6) higher in invasive local BC cases (n=182) compared to benign cases (n=22). The multivariate survival analyses revealed that the high expression of miR-7974 was significantly (P<0.05) associated with poorer relapse-free survival (RFS) and BC-specific survival (BCSS) compared to the low expression in all invasive local BC cases. Additionally, high expression of miR-7974 was linked to poorer RFS in ER+ patients, with a hazard ratio (HR) of 8.70 and a 95% confidence interval (CI) of 3.28–23.06 (P=1.37x10^-5). We also found that miR-7974 is involved in genes related to autophagy. Specifically, MAP1LC3B, a key gene involved in autophagy, was found to be a direct target of miR-7974 (P < 0.05) in MCF-7 cells. Overexpression of miR-7974 in these cells resulted in anti-proliferative effects, reducing cell growth in both in vitro and in ovo models.

Conclusion:

Our results suggest that miR-7974 could be a valuable prognostic biomarker for poor outcomes in ER+ BC, potentially improving survival predictions. The dual role of autophagy helps explain the mechanism behind poor survival, alongside the tumor-suppressive effect observed in both in vitro and in ovo models with high miR-7974 expression. Further research is needed to fully understand miR-7974’s role in autophagy regulation and its downstream effects.