LINC01410促进食管鳞状细胞癌细胞增殖和迁移的作用及其机制研究

曹飞, 俞文豪, 唐晓男, 马子冬, 常廷民, 龚亚斌, 廖明娟, 康小红

  1. 1.河南省平顶山市第一人民医院科教科，河南 平顶山 467000
    2.新乡医学院第一附属医院肿瘤科，河南 新乡 453100
    3.新乡医学院第一附属医院消化科，河南 新乡 453100
    4.上海中医药大学附属岳阳中西医结合医院肿瘤科，上海 200437
    5.上海交通大学医学院附属第九人民医院中医科，上海 200125
  • 收稿日期:2022-10-30 修回日期:2023-04-01 出版日期:2024-08-30 发布日期:2024-09-10
  • 通信作者: 康小红
  • 作者简介:曹 飞（ORCID：0009-0004-3539-3729），博士在读，主任医师，河南省平顶山市第一人民医院科教科主任。
  • 基金资助:
    国家自然科学基金(82074231);国家自然科学基金(82074268);河南省科技攻关项目(212102310209);河南省科技攻关项目(212102310705);河南省医学科技攻关项目(SB201903014);河南省医学科技攻关项目(LHGJ20200511);河南省医学科技攻关项目(LHGJ20220968)

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摘要/Abstract

摘要：

背景与目的： LINC01410是长度为647 bp的长链非编码RNA（long non-coding RNA，lncRNA），其异常表达参与多种肿瘤的发生、发展，但其对食管鳞状细胞癌（esophageal squamous cell carcinoma，ESCC）的影响研究较少。本研究旨在探讨LINC01410促进ESCC细胞增殖和迁移的作用机制，以期为防治ESCC提供生物标志物及潜在治疗靶点。方法：应用基因表达谱交互分析2（Gene Expression Profiling Interactive Analysis 2，GEPIA2）在线分析LINC01410在肿瘤与癌症基因组图谱（The Cancer Genome Atlas，TCGA）ESCC数据中的表达水平及与患者预后总生存期的关系；采用基因探针富集分析（Gene Set Enrichment Analysis，GSEA）LINC01410可能调控的信号转导通路。收集2020年1月—2021年12月在新乡医学院第一附属医院、平顶山市第一人民医院胸外科行根治性手术的ESCC患者新鲜肿瘤组织标本62例，并选取癌旁组织作为对照。本项目经医院伦理委员会批准（新乡医学院第一附属医院，编号：2018036；平顶山市第一人民医院，编号：2019-018）。本研究采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）检测LINC01410的表达水平并分析其与临床病理学参数的关系。应用LV-NC、LV-over/LINC01410转染EC109细胞并建立稳定过表达细胞株EC109/NC、EC109/OE，采用shRNA-NC、shRNA-LINC01410转染EC9706细胞并建立稳定敲降细胞株EC9706/NC、EC9706/KD；应用RTFQ-PCR检测LINC01410的相对表达量；采用噻唑蓝比色法[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，MTT]实验和克隆形成实验检测细胞增殖；采用transwell实验检测细胞迁移能力；采用双荧光素酶报告基因实验检测LINC01410对T细胞因子/淋巴增强因子1（T cell factor/lymphoid enhancer factor 1，TCF/LEF1）启动子活性的影响；采用蛋白质印迹法（Western blot）检测Wnt/β-连环蛋白（β-catenin）信号转导通路、上皮-间质转化（epithelial-mesenchymal transition，EMT）信号转导通路相关蛋白表达水平。结果：GEPIA2分析显示，在ESCC组织中LINC01410表达水平显著高于食管正常组织，两组相比差异有统计学意义（P<0.05）。生存分析显示，高表达LINC01410的ESCC患者生存率显著低于低表达LINC01410的患者（P=0.042）。RTFQ-PCR检测结果显示，在ESCC组织中LINC01410表达水平显著高于配对癌旁组织；LINC01410高表达与肿瘤浸润程度、淋巴结转移及TNM分期显著相关（P<0.01）；与食管正常上皮细胞HEEC相比，LINC01410在EC109、EC9706细胞中显著高表达（P<0.05）；LINC01410在EC109/OE细胞中显著高表达（P<0.01），而在EC9706/KD细胞中显著下调（P<0.01）。MTT结果显示，EC109/OE细胞48 h时细胞活性显著高于EC109/NC细胞（P<0.01）；EC9706/KD细胞48 h时细胞活性显著低于EC9706/NC（P<0.01）。细胞克隆形成实验结果显示，与EC109/NC细胞相比，EC109/OE细胞克隆数显著增多；与EC9706/NC细胞相比，EC9706/KD细胞克隆数明显减少（P<0.01）。Transwell迁移实验结果显示，EC109/OE细胞株迁移的细胞数显著多于EC109/NC细胞（P<0.01），而EC9706/KD细胞迁移的细胞数显著少于EC9706/NC细胞（P<0.01）。GSEA分析显示，Wnt/β-catenin、EMT等信号转导通路显著富集在高表达LINC01410的ESCC组织中。双荧光素酶报告基因实验结果示，EC109/OE细胞中TCF/LEF1启动子活性显著高于EC109/NC细胞，EC9706/KD细胞中TCF/LEF1启动子活性显著低于EC9706/NC细胞（P<0.01）。Western blot检测结果显示，与EC109/NC细胞相比，EC109/OE细胞中E-cadherin蛋白表达下调，而N-cadherin、β-cantenin、cyclin D1、c-Myc等蛋白显著上调；与EC9706/NC细胞相比，E-cadherin蛋白表达上调，而N-cadherin、β-cantenin、cyclin D1、c-Myc等蛋白显著下调。结论：LINC01410具有促进ESCC细胞增殖和迁移的作用，其机制可能与激活EMT、Wnt/β-catenin等信号转导通路有关。

关键词: 食管鳞癌, 长链非编码RNA-LINC01410, 增殖, 迁移, Wnt/β-catenin信号转导通路

Abstract:

Background and purpose: Long non-coding RNA (lncRNA) LINC01410, with a length of 647 bp, participates in a variety of tumor biological processes. However, the role and mechanism of LINC01410 involved in esophageal squamous cell carcinoma (ESCC) remain unclear. This study aimed to explore the potential mechanism of LINC01410 promoting ESCC proliferation and invasion, to provide a potential prognostic indicator and therapeutic target for individuals with ESCC. Methods: Gene Expression Profiling Interactive Analysis 2 (GEPIA2) databases were used to analyze the expression of LINC01410 and overall survival in esophageal squamous cell carcinoma data set in the Cancer Genome Atlas (TCGA). Gene Set Enrichment Analysis (GSEA) was performed to identify the underlying signaling pathways involved in the biological effects of LINC01410 in ESCC. A total of 62 pairs of ESCC tissues and paracancerous tissues from ESCC patients who underwent radical surgery in the Department of Thoracic Surgery at the First Affiliated Hospital of Xinxiang Medical College and the First People's Hospital of Pingdingshan City from January 2020 to December 2021 were collected. This project has been approved by the Hospital Ethics Committee (First Affiliated Hospital of Xinxiang Medical College, No. 2018036; First People's Hospital of Pingdingshan City, No. 2019-018). The expression of LINC01410 in ESCC tissues was detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). We transfected EC109 cells with LV-NC or LV-over/LINC01410 and EC9706 cells with shRNA-NC or shRNA-LINC01410. Stable transfected cells (EC109/NC, EC109/OE, EC9706/NC and EC9706/KD) were selected in primary cell culture medium containing puromycin. The expression of LINC01410 was detected by RTFQ-PCR. The impact of LINC01410 on ESCC cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. The effect of LINC01410 on ESCC cell invasion was detected by transwell migration assay. T cell factor/lymphoid enhancer factor 1 (TCF/LEF1) luciferase reporter assay was performed to validate the effect of LINC01410 on the activity of canonical Wnt/β-catenin signaling pathway. The expressions of Wnt/β-catenin and epithelial-mesenchymal transition (EMT) signal pathway related proteins in ESCC cells were detected by Western blot. Results: By analyzing the LINC01410 expression from ESCC samples in TCGA by GEPIA2, we found LINC01410 was consistently increased in ESCC tumors compared with normal tissues (P<0.05), and high LINC01410 expression was associated with poorer overall survival (OS). RTFQ-PCR assay showed that expressions of LINC01410 were higher in esophageal cancer tissues and esophageal cancer cells (EC109 and EC9706) than in precancerous tissues and HEEC cells (P<0.05). The expression level of LINC01410 was significantly correlated with invasion range, lymph node metastasis and TNM stage in ESCC patients (P<0.01). LINC01410 expression was also upregulated in EC109/OE, however the expression of LINC01410 in EC9706/KD was decreased (P<0.01). MTT assay showed overexpression of LINC01410 increased the viability of EC109 cells, while knockdown of LINC01410 decreased the viability of EC9706 cells (P<0.01). Colony formation assay indicated that overexpression of LINC01410 enhanced the clonogenic ability of ESCC cells, while knockdown of LINC01410 reduced colony formation (P<0.01). Transwell migration assay showed that LINC01410 overexpression drastically increased the number of migratory cells, while silencing of LINC01410 suppressed the migration in EC9706 cells (P<0.01). GSEA revealed that Wnt/β-catenin and EMT pathways were significantly enriched in ESCC samples with a high level of LINC01410. TCF/LEF1 luciferase reporter assay showed higher levels of Wnt-dependent activities were observed in EC109/OE cells, whereas silenced LINC01410 in EC9706 cells led to contrary results (P<0.01). Western blot analysis showed that overexpression of LINC01410 in EC109 cell significantly increased the expression levels of N-cadherin, β-catenin, cyclin D1, c-Myc and decreased E-cadherin expression, while knockdown LINC01410 resulted in opposite results. Conclusion: LINC01410 promotes proliferation and metastasis of ESCC, which might be caused by activation of Wnt/β-catenin and EMT signaling pathways.

Key words: Esophageal squamous cell carcinoma, Long non-coding RNA, LINC01410, Proliferation, Invasion, Wnt/β-catenin signaling pathway

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