[摘 要] 目的：通过筛选在食管鳞状细胞癌（ESCC）患者血清外泌体（Exo）中高表达的微小RNA（miRNA）并分析其与患者临 床病理特征的关系，探讨Exo来源的miRNA是否具有成为ESCC临床辅助诊断标志物的潜力。方法：采集2021年12月至2023 年6月期间在河北医科大学第四医院收治的45例ESCC初诊患者和50例健康受试者的血清及相关临床资料，分别作为ESCC组 和对照组。用基因表达综合数据库（GEO）和qPCR法筛选、鉴定出ESCC患者血清表达升高的候选miRNA-miR-1246，用受试者 工作特征曲线分析血清miR-1246对ESCC的诊断效能，Logistic回归分析其与ESCC患者临床特征进展的关系，χ2 检验分析其与 ESCC患者临床病理特征的关系。分离纯化受试者血清中的Exo并进行表征验证，qPCR检测Exo中miR-1246的表达。常规培养 ESCC KYSE150和KYSE30细胞，用Lipofectamine 2000分别将mimics-NC、miR-1246 mimics转染至KYSE150细胞，将inhibitorNC和miR-1246 inhibitor转染至KYSE30细胞，分别记为mimics-NC、miR-1246mimics、 inhibitor-NC和miR-1246-inhibitor组。用 mimics-NC 和 miR-1246 mimics 组 KYSE150 细胞来源的 Exo 处理 KYSE150 和 KYSE30 细胞。用 CCK-8 法、划痕愈合实验、 Transwell小室实验分别检测各组细胞的增殖、迁移和侵袭能力。WB法检测Exo标志物及各组细胞中上皮间皮转化相关蛋白及 Tet甲基胞嘧啶双加氧酶2（TET2）和细胞黏附分子1（CADM1）蛋白的表达。双萤光素酶报告基因实验验证miR-1246与TET2和 CADM1的靶向结合关系。结果：生物信息学筛选ESCC患者血清中差异表达最为显著的miRNA为miR-1246。试验提取患者 的血清Exo符合典型Exo表征。Ⅰ~Ⅱ期ESCC患者血清Exo-miR-1246表达水平显著高于健康受试者（P < 0.01）；Ⅲ~Ⅳ期ESCC 患者的血清Exo-miR-1246水平明显高于Ⅰ~Ⅱ期患者（P < 0.01）。ROC曲线分析表明，血清中的Exo-miR-1246对ESCC有较高 的辅助鉴别诊断价值（P < 0.05），并且Exo-miR-1246对ESCC患者临床进展的辅助诊断效能高于CEA与SCC-Ag（P < 0.05），三者 联合检测会进一步提高辅助诊断患者分期效能（P < 0.01）。Exo-miR-1246可能是ESCC患者临床进展的独立危险因素（P < 0.05）。 血清Exo-miR-1246表达水平与ESCC的T分期、N分期和临床分期有关联（P < 0.01）。过表达miR-1246可促进ESCC细胞增殖、 迁移、侵袭、上皮间质转化和抑制凋亡而抑制miR-1246则相反。数据库数据分析发现，TET2和CADM1是miR-1246的靶基因， 双萤光素酶报告基因实验证实miR-1246可直接与TET2和CADM1 mRNA结合并抑制其表达（P < 0.01）。用过表达miR-1246的 细胞来源的Exo处理KYSE150和KYSE30细胞与在其中过表达miR-1246的作用一致。结论: Exo来源的miR-1246具有成为 ESCC临床辅助诊断标志物的潜力，其可能通过调控TET2和CADM1的表达水平来影响ESCC的发生发展。

[Abstract] Objective: To screen for microRNAs (miRNAs) highly expressed in the serum exosomes (Exo) of esophageal squamous cell carcinoma (ESCC) patients and analyze their relationship with the clinicopathological characteristics of the patients, and to explore the potential of Exo-derived miRNAs as clinical auxiliary diagnostic markers for ESCC. Methods: Serum and relevant clinical data of 50 healthy subjects and 45 newly diagnosed ESCC patients admitted to the Fourth Hospital of Hebei Medical University between December 2021 and June 2023 were collected, serving as the control group and the ESCC group respectively. The Gene Expression Omnibus (GEO) database and qPCR were used to screen and identify the candidate miRNA for increased expression in the serum of ESCC patients-miR-1246. The diagnostic efficacy of serum miR-1246 for ESCC was analyzed by the receiver operating characteristic curve. The relationship between miR-1246 and the clinical feature progression of ESCC patients was analyzed by Logistic regression, and the relationship between miR-1246 and the clinicopathological characteristics of ESCC patients was analyzed by the χ2 test. Exosomes in the serum of the subjects were isolated, purified and characterized for verification. The expression of miR-1246 in Exo was detected by qPCR. ESCC KYSE150 and KYSE30 cells were routinely cultured. mimics-NC and miR-1246 mimics were transfected respectively into KYSE150 cells using Lipofectamine 2000. Inhibitor-NC and miR-1246 inhibitor were transfected into KYSE30 cells, which were respectively denoted as the minics-NC, miR-1246 mimics, inhibitor-NC and miR-1246-inhibitor groups. KYSE150 and KYSE30 cells were treated with Exo derived from KYSE150 cells in the mimics-NC and miR-1246 mimics groups. The proliferation, migration and invasion abilities of cells in each group were detected by the CCK-8 assay, scratch wound healing assay and Transwell chamber assay respectively. The expressions of Exo markers, epithelial-mesenchymal transition-related proteins, TET family methylcytosine dioxygenase 2 (TET2) and cell adhesion molecule 1 (CADM1) proteins in each group of cells were detected by WB assay. The targeting binding relationship between miR-1246 and TET2 and CADM1 was verified by the dual-luciferase reporter gene assay. Results: Bioinformatics screening showed that the miRNA with the most significant differential expression in the serum of ESCC patients was miR-1246. The serum Exo extracted from the patients conformed to the typical Exo characteristics. The expression level of serum Exo-miR-1246 in ESCC patients at stages Ⅰ-Ⅱ was significantly higher than that in healthy subjects (P < 0.01); the level of serum Exo-miR-1246 in ESCC patients at stages Ⅲ-Ⅳ was significantly higher than that in patients at stages Ⅰ-Ⅱ (P < 0.01). ROC curve analysis showed that Exo-miR-1246 in serum had a high value for auxiliary differential diagnosis of ESCC (P < 0.05), and the auxiliary diagnostic efficacy of Exo-miR-1246 for the clinical progression of ESCC patients was higher than that of CEA and SCC-Ag (P < 0.05). The combined detection of the three could further improve the efficacy of auxiliary diagnosis of patient staging (P < 0.01). Exo-miR-1246 might be an independent risk factor for the clinical progression of ESCC patients (P < 0.05). The expression level of serum Exo-miR-1246 was associated with the T-stage, N-stage and clinical stage of ESCC (P < 0.01). Overexpression of miR-1246 could promote the proliferation, migration, invasion, epithelial-mesenchymal transition and inhibit apoptosis of ESCC cells, while inhibition of miR-1246 had the opposite effect. Database data analysis found that TET2 and CADM1 were the target genes of miR-1246. The dual-luciferase reporter gene assay confirmed that miR-1246 could directly bind to TET2 and CADM1 mRNA and inhibit their expressions (P < 0.01). Treatment of KYSE150 and KYSE30 cells with Exo derived from cells overexpressing miR-1246 had the same effect as overexpressing miR-1246 in these cells. Conclusion: Exo-derived miR-1246 has the potential to be a clinical auxiliary diagnostic marker for ESCC. It may affect the occurrence and development of ESCC by regulating the expression levels of TET2 and CADM1.

河北省自然科学基金（No. H201806115，No. H2024206074）；河 北 医科大学“十 四 五”临 床 医 学 创 新 研 究 团 队 支 持 计划 （No. 2022LCTD-B43）；河北省政府资助临床医学优秀人才培养项目（No. ZF2025191）；河北省卫生健康创新专项（No. 22377791D）；河北省医学 科学研究重点课题计划（20180483）