Fig. 2

Study design and identification of plasma miRNA candidates. (a) Study design to identify novel candidate miRNAs with reduced levels in patient plasma as potential therapeutic targets for ESCC. The workflow included: (1) systematic in silico screening and experimental selection of candidate miRNAs; (2) large-scale qRT-PCR validation of plasma miR-3619-5p levels in ESCC patients (n = 94) and healthy volunteers (n = 81); (3) assessment of associations between plasma levels and clinicopathological features or survival; and (4) functional validation through in vitro and in vivo experiments. (b) The expression levels of miR-3619-5p were analyzed in normal human organs and ESCC cell lines. High expression of miR-3619-5p was observed in the skeletal muscle, heart, stomach, and lung. In contrast, 75% of ESCC cell lines exhibited lower miR-3619-5p expression compared to normal esophageal mucosa. (c) Quantitative analysis of plasma miR-3619-5p levels in clinical samples. Plasma RNA was isolated from peripheral blood of ESCC patients and healthy volunteers. Levels of miR-3619-5p were measured by TaqMan qRT-PCR and normalized to cel-miR-39. Statistical comparison using the Mann–Whitney U test revealed significantly reduced plasma miR-3619-5p levels in ESCC patients (P = 0.0002), suggesting potential utility as a non-invasive biomarker. (d) Kaplan–Meier survival analysis based on plasma miR-3619-5p expression. Prognostic analysis demonstrated that low plasma levels of miR-3619-5p were significantly correlated with poorer overall survival rates (P = 0.0471). Additionally, low plasma levels of miR-3619-5p were significantly correlated with poorer recurrence-free survival rates in ESCC patients (P = 0.0378).