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