IntroductionGlobally, esophageal cancer ranks sixth in mortality rates, contributing significantly to cancer death statistics1. Esophageal cancer is typically classified into two histological types: adenocarcinoma and squamous cell carcinoma2. In Western countries, adenocarcinoma is the predominant form of esophageal cancer. In contrast, within the “esophageal cancer belt,” a region spanning from northern Iran through Central Asia to north-central China, esophageal squamous cell carcinoma (ESCC) accounts for roughly 90% of all cases1. In recent years, remarkable advancements have been seen in chemotherapy, radiotherapy, surgical procedures, and perioperative management for ESCC. Despite advancements in treatment, the 5-year survival rate for individuals with ESCC continues to hover around 20%3. To enhance patient outcomes, many studies have focused on elucidating the molecular mechanisms of tumorigenesis in ESCC, as well as identifying potential clinical biomarkers and therapeutic molecular targets. However, for ESCC there are still only a few early diagnostic biomarkers and therapeutic targets4,5.Discovered in 1993, microRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression by destabilizing mRNA and inhibiting its translation. Numerous studies have revealed altered miRNA expression associated with the onset and development of diverse diseases, including cancer6. In particular, several miRNAs have been shown to maintain their stability and detectability in plasma and serum by being encapsulated within exosomes and binding to proteins such as Argonaute2 and HDL. These interactions provide protection, preventing the miRNAs from being degraded by endogenous ribonucleases7,8,9,10. Furthermore, some of these blood miRNAs have been reported not only to be derived from tumor cell lysis but also actively secreted as mediators of intercellular communication8,11,12,13. Over the past decade, numerous blood-derived miRNAs, including those examined in this study, have emerged as potential biomarkers for detecting cancer, monitoring tumor progression, and predicting prognosis as well as chemoresistance14,15,16,17.Kosaka and collaborators proposed a new theory based on findings from their in vitro research18. Over the past decade, numerous blood-derived miRNAs, including those examined in this study, have emerged as potential biomarkers for detecting cancer, monitoring tumor progression, and predicting prognosis as well as chemoresistance. However, as the disease advances, the compensatory mechanism of healthy cells weakens, contributing to cancer progression.Our previous research revealed a decline in specific tumor-suppressor miRNAs in the blood as cancer advances14,15,17,19. We suggested that the levels of these miRNAs in the blood might indicate the capacity of adjacent healthy cells to transfer tumor-suppressor miRNAs to cancer cells. As a result, specific tumor-suppressor miRNAs in the bloodstream may serve as valuable biomarkers for detecting malignant cancers, evaluating prognosis and chemosensitivity, and acting as potential targets for nucleic acid-based therapies in digestive tract cancers17,20,21,22.As part of this research, we concentrated on the tumor-suppressor miR-3619-5p, identified through a comprehensive analysis of the NCBI and miRNA databases. This miRNA, which targets the proviral insertion site in Moloney murine leukemia virus 1 (PIM1), was observed to be diminished in the plasma of ESCC patients. In conclusion, our results showed that lower plasma levels of miR-3619-5p were strongly linked to advanced pathological stages and recognized as an independent prognostic indicator. Enhanced expression of miR-3619-5p in ESCC cells suppressed cell proliferation by inducing apoptosis or causing G1/S arrest in a TP53-dependent manner, while also impairing cell migration and invasion. Overexpression of miR-3619-5p in ESCC cells directly suppressed PIM1 in vitro. In mice, increased plasma 3619-5p levels following subcutaneous injection significantly inhibited tumor growth, with downregulation of PIM1. Overall, our findings indicated that reduced blood levels of miR-3619-5p are linked to ESCC progression and poor prognosis, highlighting the possibility that miR-3619-5p could be a potential target for nucleic acid therapies targeting PIM1 in ESCC patients (Fig. 1).Fig. 1Graphical abstract. In healthy persons, the plasma level of miR-3619-5p are sufficiently supplied. However, as esophageal squamous cell carcinoma (ESCC) progresses, the plasma level of miR-3619‐5p is decreasing. This dynamics of miR-3619‐5p could be the promising biomarker to detect cancer and predict prognoses of ESCC patients. Moreover, the overexpression of miR-3619‐5p suppresses proviral insertion site in Moloney murine leukemia virus 1 (PIM1) expression by inhibiting stability and translation of mRNA. Overexpression of miR-3619‐5p enhances the tumor-killing activity by suppressing PIM1 expression. Low blood levels of miR-3619‐5p are associated with ESCC progression and poor prognoses and could be a target of nucleic acid therapy in ESCC patients.Full size imageResultsSelection of plasma MiRNA candidates based on a systematic review of the NCBI databaseFrom the 2,600 miRNA candidates available in miRBase, we identified 25 miRNAs that exhibit low expression levels in ESCC tissues, possess tumor-suppressive properties, and have not yet been reported as biomarkers in body fluids. Following the application of exclusion criteria, five miRNAs (miR-3619-5p, −3178, −1182, −637, and − 564) were selected based on their detectable signals in samples from healthy individuals (Supplementary Fig. 1). Among the five candidate miRNAs, miR-3619-5p was chosen in this study, based on its tumor-suppressive function, detectable levels in healthy plasma, and lack of prior reports as a circulating biomarker in ESCC23,24.Study design to find novel biomarker MiRNAs in the plasma of ESCC patientsThis study was structured as outlined below. (1) Plasma miR-3619-5p levels were analyzed to examine their correlations with clinicopathological characteristics and prognostic outcomes in ESCC patients; (2) an evaluation of the potential anti-tumor effects of miR-3619-5p overexpression in ESCC cells was undertaken in vitro; and (3) an assessment of its tumor-suppressive role was carried out in an in vivo tumor model (Fig. 2a).Fig. 2Study 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).Full size imageLevels of miR-3619-5p in normal tissues and ESCC cell linesWe examined miR-3619-5p expression levels in normal human tissues and ESCC cell lines to determine its tissue distribution and potential cancer-specific downregulation. Total RNA was extracted from commercially available normal human tissue samples (stomach, thymus, prostate, skeletal muscle, kidney, spleen, intestine, pancreas, heart, lung, colon, testis, liver, thyroid, brain, and esophagus) and sixteen ESCC cell lines (TE1, TE2, TE4, TE5, TE6, TE8, TE9, TE10, TE11, TE13, TE14, TE15, KYSE70, KYSE150, KYSE170, and KYSE790). miR-3619-5p levels were quantified using TaqMan-based quantitative reverse transcription PCR (qRT-PCR), and normalized to U6 small nuclear RNA (RNU6B) using the 2^−ΔΔCt method. All measurements were conducted in triplicate (n = 3 biological replicates per sample).As depicted in Fig. 2b, miR-3619-5p exhibited relatively high expression in the skeletal muscle, heart, stomach, and lung among the tested normal tissues, suggesting its physiological role in multiple organs. In contrast, its expression was consistently downregulated in ESCC cell lines when compared to normal esophageal mucosa. Specifically, 12 out of 16 (75%) ESCC cell lines showed more than 50% reduction in miR-3619-5p levels.Comprehensive validation of plasma miR-3619-5p levels in ESCC patientsWe initiated our analysis by validating the findings in a large-scale study. Plasma miR-3619-5p was successfully detected in all 94 ESCC patients and 81 healthy volunteers. Notably, plasma miR-3619-5p levels were significantly reduced in ESCC patients compared to healthy controls (P