MicroRNAs (miRNAs) are involved in the development of human malignancies, and cells have the ability to secrete these molecules into extracellular compartments. Thus, cell-free miRNAs (circulating miRNAs) can potentially be used as biomarkers to evaluate pathophysiological changes. Although circulating miRNAs have been proposed as potential noninvasive tumor biomarkers for diagnosis, prognosis, and response to therapy, their routine application in the clinic is far from being achieved. This review focuses on the recent progress regarding the value of circulating miRNAs as noninvasive biomarkers, with specific consideration of their relevant clinical applications. In addition, we provide an in-depth analysis of the technical challenges that impact the assessment of circulating miRNAs. We also highlight the significance of integrating circulating miRNAs with the standard laboratory biomarkers to boost sensitivity and specificity. The current status of circulating miRNAs in clinical trials as tumor biomarkers is also covered. These insights and general guidelines will assist researchers in experimental practice to ensure quality standards and repeatability, thus improving future studies on circulating miRNAs. SIGNIFICANCE STATEMENT Our review will boost the knowledge behind the inconsistencies and contradictory results observed among studies investigating circulating miRNAs. It will also provide a solid platform for better-planned strategies and standardized techniques to optimize the assessment of circulating cell-free miRNAs.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor–positive (ER+) BC, we hypothesized that 17β-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17β-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7’s miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17β-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.