吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (5): 1217-1226.doi: 10.13481/j.1671-587X.20230515

• 基础研究 • 上一篇    

巨噬细胞外泌体lncRNA HULC对肝癌细胞迁移、侵袭和转移的影响及其机制

董勇,徐菱遥,华静,梁晗,刘东亚,赵俊波,孙正路,程序,魏书堂()   

  1. 河南大学第一附属医院消化内科,河南 开封 475100
  • 收稿日期:2022-12-27 出版日期:2023-09-28 发布日期:2023-10-26
  • 通讯作者: 魏书堂 E-mail:13460772602@163.com
  • 作者简介:董 勇(1988-),男,河南省民权市人,主治医生,医学硕士,主要从事肝胆胰腺临床治疗方面的研究。
  • 基金资助:
    河南省卫健委医学科技攻关计划联合共建项目(LHGJ20200539)

Effect of macrophage exosomal lncRNA HULC on migration, invasion,and metastasis of hepatocellular carcinoma cells and its mechanism

Yong DONG,Lingyao XU,Jing HUA,Han LIANG,Dongya LIU,Junbo ZHAO,Zhenglu SUN,Cheng CHENG,Shutang WEI()   

  1. Department of Gastroenterology,First Affiliated Hospital,Henan University,Kaifeng 475100,China
  • Received:2022-12-27 Online:2023-09-28 Published:2023-10-26
  • Contact: Shutang WEI E-mail:13460772602@163.com

摘要:

目的 探讨巨噬细胞外泌体长链非编码RNA(lncRNA)肝癌高表达转录本(HULC)在肝细胞癌(HCC)转移中的作用,并阐明其作用机制。 方法 分离小鼠骨髓单核细胞,采用佛波酯诱导分化为骨髓源巨噬细胞(BMDM),并用白细胞介素4(IL-4)将BMDM诱导分化为M2巨噬细胞,即肿瘤相关巨噬细胞(TAM)。差速离心法收集M2巨噬细胞外泌体(TAM-exos)。在TAM中分别转染lncRNA HULC-siRNA或NC质粒后提取各组TAM分泌的外泌体(lncRNA HULC-siRNA-exos或NC-exos)。将HepG2细胞按照实验目的分为对照组和TAM-exos组;NC-exos组和lncRNA HULC-siRNA-exos组;lncRNA HULC-siRNA-exos组和lncRNA HULC-siRNA-exos+SKL2001组。给予相应处理后,Transwell小室实验检测HepG2细胞的迁移和侵袭细胞数,实时荧光定量PCR(RT-qPCR)法检测HepG2细胞中lncRNA HULC表达水平,Western blotting法检测HepG2细胞中Wnt3A、β-连环蛋白(β-catenin)、c-Myc和Cyclin D1蛋白表达水平。构建裸鼠原位肝癌移植模型,分为对照组、NC-exos组、TAM-exos组和lncRNA HULC-siRNA-exos组,检测M2巨噬细胞外泌体lncRNA HULC作用后裸鼠原位移植瘤肺转移灶数。 结果 TAM-exos符合外泌体的形态和生物学特征。与对照组比较,TAM-exos组HepG2细胞的迁移和侵袭细胞数及细胞中lncRNA HULC、Wnt3A、β-catenin、c-Myc和Cyclin D1蛋白表达水平升高(P<0.05)。与NC-exos组比较,lncRNA HULC-siRNA-exos组HepG2细胞的迁移和侵袭细胞数及细胞中lncRNA HULC、Wnt3A、β-catenin、c-Myc和Cyclin D1蛋白表达水平降低(P<0.05)。与lncRNA HULC-siRNA-exos组比较,lncRNA HULC-siRNA-exos+SKL2001组HepG2细胞的迁移和侵袭细胞数及细胞中Wnt3A、β-catenin、c-Myc和Cyclin D1蛋白表达水平升高(P<0.05)。裸鼠实验,M2巨噬细胞外泌体lncRNA HULC作用后,TAM-exos组裸鼠原位移植瘤肺转移灶数较对照组增多(P<0.05);M2巨噬细胞外泌体lncRNA HULC作用后,lncRNA HULC-siRNA-exos组裸鼠原位移植瘤肺转移灶数较TAM-exos组和NC-exos组减少(P<0.05)。 结论 TAM外泌体lncRNA HULC具有促进HCC细胞侵袭和转移的作用,其作用机制可能与激活Wnt信号通路有关。

关键词: 肝细胞肿瘤, 外泌体, 肿瘤相关巨噬细胞, 细胞迁移, 细胞侵袭

Abstract:

Objective To discuss the effect of macrophage-derived extracellular vesicle long non-code RNA(lncRNA) highly up-regulated in liver cancer(HULC) on the metastasis of hepatocellular carcinoma (HCC),and to elucidate its mechanism. Methods The bone marrow mononuclear cells were isolated from the mice and differentiated into bone marrow-derived macrophages (BMDM) through phorbol myristate acetate induction. Interleukin-4 (IL-4) was used to induce the M2 macrophage polarization,regarded as tumor-associated macrophages(TAM).The TAM-derived extracellular vesicles (referred to as TAM-exos) were collected using differential centrifugation. RNA interference was used to transfect TAM with lncRNA HULC siRNA or negative control (NC) plasmids, and the extracellular vesicles secreted by TAM in various groups were extracted (referred to as lncRNA HULC-siRNA-exos and NC-exos). The HepG2 cells were divided into control group and TAM-exos group; NC-exos group and lncRNA HULC-siRNA-exos group; lncRNA HULC-siRNA-exos group and lncRNA HULC-siRNA-exos+SKL2001 group according to the purpose of the experiment. After the corresponding treatments, Transwell champer assay was used to detect the numbers of migration and invasion HepG2 cells in various groups; real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression level of lncRNA HULC in the HepG2 cells in various groups; Western blotting method was used to detect the expression levels of Wnt3A, β-catenin, c-Myc, and Cyclin D1 in the HepG2 cells in various groups. The nude mouse orthotopic liver cancer transplantation model was established, and the mice were divided into control group, NC-exos group, TAM-exos group, and lncRNA HULC-siRNA-exos group. The numbers of lung metastatic lesions in the nude mice in various groups after treatment with M2 macrophage-derived extracellular vesicles containing lncRNA HULC were detected. Results The TAM-exos displayed the morphology and biological characteristics of extracellular vesicles. Compared with control group, the numbers of migration and invasion HepG2 cells in TAM-exos group were increased(P<0.05), and the expression levels of lncRNA HULC, Wnt3A, β-catenin, c-Myc, and Cyclin D1 proteins in the HepG2 cells in TAM-exos group were increased (P<0.05). Compared with NC-exos group, the numbers of migration and invasion HepG2 cells in lncRNA HULC-siRNA-exos group were decreased,and the expression levels of lncRNA HULC, Wnt3A, β-catenin, c-Myc, and Cyclin D1 proteins in the HepG2 cells were decreased (P<0.05). Compared with lncRNA HULC-siRNA-exos group, the numbers of migration and invasion HepG2 cells in lncRNA HULC-siRNA-exos+SKL2001 group were increased(P<0.05),and the expression levels of Wnt3A, β-catenin, c-Myc, and Cyclin D1 proteins in the HepG2 cells were increased (P<0.05). The nude mouse experiment results showed that compared with control group, the number of lung metastatic lesions of the nude mice in TAM-exos group was increased (P<0.05); compared with TAM-exos group and the NC-exos group, the number of lung metastatic lesions of the nude mice in lncRNA HULC-siRNA-exos group was decreased (P<0.05). Conclusion The TAM-derived extracellular vesicle lncRNA HULC promotes the invasion and metastasis of the HCC cells, and its mechanism may be associated with the activation of the Wnt signaling pathway.

Key words: Hepatocellular carcinoma, Extracellular vesicles, Tumor-associated macrophages, Cell migration, Cell invasion

中图分类号: 

  • R735.7