食管鳞状细胞癌患者血清外泌体miR-1246的表达及其临床意义  

Expression and clinical significance of serum exosome miR-1246 in patients with esophageal squamous cell carcinoma

作　　者：赵薇 崔雯瑄 黄焙炫 尚晓雅 王振达 杜彦艳[1] 赵红峥[1] 焦文静[1] 马鸣[1] ZHAO Wei;CUI Wenxuan;HUANG Beixuan;SHANG Xiaoya;WANG Zhenda;DU Yanyan;ZHAO Hongzheng;JIAO Wenjing;MA Ming(Department of Clinical Laboratory,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,Hebei,China)

机构地区：[1]河北医科大学第四医院检验科,河北石家庄050011

出　　处：《中国肿瘤生物治疗杂志》2025年第2期176-188,共13页Chinese Journal of Cancer Biotherapy

基　　金：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)。

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,χ^(2)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inhibitor-NC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SCCObjective: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 χ^(2)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 c

关 键 词：外泌体 miR-1246 食管鳞状细胞癌 Tet甲基胞嘧啶双加氧酶2 细胞黏附分子1 临床意义 

分 类 号：R735.1[医药卫生—肿瘤] R730.43[医药卫生—临床医学]