Recent studies have demonstrated that long non-coding RNAs (lncRNAs) were present in the blood of cancer patients and have shown great potential as powerful and non-invasive tumor markers. However, little is known about the value of lncRNAs in the diagnosis of esophageal squamous cell carcinoma (ESCC). We hypothesized that ESCC-related lncRNAs might be released into the circulation during tumor initiation and could be utilized to detect and monitor ESCC. Ten lncRNAs (HOTAIR, AFAP1-AS1, POU3F3, HNF1A-AS1, 91H, PlncRNA1, SPRY4-IT1, ENST00000435885.1, XLOC_013104 and ENST00000547963.1) which previously found to be differently expressed in esophageal cancer were selected as candidate targets for subsequent circulating lncRNA assay. A four-stage exploratory study was conducted to test the hypothesis: (1) optimization of detected method to accurately and reproducibly measure ESCC-related lncRNAs in plasma and serum; (2) evaluation of the stability of circulating lncRNAs in human plasma or serum; (3) exploration the origin of ESCC-related lncRNAs in vitro and in vivo; (4) evaluation the diagnostic power of circulating lncRNAs for ESCC. ESCC-related lncRNAs were detectable and stable in plasma of cancer patients, and derived largely from ESCC tumor cells. Furthermore, plasma levels of POU3F3, HNF1A-AS1 and SPRY4-IT1 were significantly higher in ESCC patients compared with normal controls. By receiver operating characteristic curve (ROC) analysis, among the three lncRNAs investigated, plasma POU3F3 provided the highest diagnostic performance for detection of ESCC (the area under the ROC curve (AUC), 0.842; p < 0.001; sensitivity, 72.8%; specificity, 89.4%). Moreover, use of POU3F3 and SCCA in combination could provide a more effective diagnosis performance (AUC, 0.926, p < 0.001, sensitivity, 85.7%; specificity, 81.4%). Most importantly, this combination was effective to detect ESCC at an early stage (80.8%). Plasma POU3F3 could serve as a potential biomarker for diagnosis of ESCC, and the combination of POU3F3 and SCCA was more efficient for ESCC detection, in particular for early tumor screening.

This study aimed to investigate the role of ferroptosis-related long non-coding RNA CASC9 in the carcinogenic process of esophageal squamous cell carcinoma and evaluate its clinical diagnostic and prognostic value. LncRNA microarray screening was performed to identify differentially expressed genes in ESCC tissues. CASC9 expression was validated in 150 paired ESCC tissues, precancerous lesions, and cell lines by qRT-PCR. The correlation between CASC9 expression and clinicopathological characteristics as well as survival outcomes was analyzed. In vitro functional assays were conducted to examine the effects of CASC9 on ESCC cell proliferation, apoptosis, invasion, and migration. The role of CASC9 in ferroptosis was explored by detecting GSH, MDA, and ROS levels. CASC9 was significantly overexpressed in 92.7% of ESCC tissues (mean 11-fold upregulation), showing stepwise elevation during disease progression (normal mucosa→LGIN→HGIN→invasive carcinoma); High CASC9 expression correlated significantly with depth of invasion (P = 0.001) and clinical stage (P = 0.033), serving as an independent risk factor for overall survival (HR = 1.550, P = 0.026); Plasma CASC9 levels were significantly elevated in ESCC patients and markedly decreased at 14 and 30 days postoperatively. CASC9 knockdown markedly suppressed ESCC cell proliferation, invasion, and migration while promoting apoptosis. Reduced GSH and MDA/ROS accumulation indicate that CASC9 regulates ESCC progression via the ferroptosis pathway. In combination with clinicopathological features, a nomogram based on the gene signature presented strong predictive power and risk stratification for ESCC. CASC9 functions as an oncogenic lncRNA that promotes ESCC progression by modulating ferroptosis, representing a promising early diagnostic biomarker, prognostic predictor, and therapeutic target.