Hepatocellular Carcinoma Cell-Derived Exosomal miR-21-5p Induces Macrophage M2 Polarization by Targeting RhoB

Abstract

M2-like polarized tumor-associated macrophages (TAMs) are the major component of infiltrating immune cells in hepatocellular carcinoma (HCC), which have been proved to exhibit significant immunosuppressive and pro-tumoral effects. However, the underlying mechanism of the tumor microenvironment (TME) educating TAMs to express M2-like phenotypes is still not fully understood. Here, we report that HCC-derived exosomes are involved in intercellular communications and exhibit a greater capacity to mediate TAMs' phenotypic differentiation. In our study, HCC cell-derived exosomes were collected and used to treat THP-1 cells in vitro. Quantitative polymerase chain reaction (qPCR) results showed that the exosomes significantly promoted THP-1 macrophages to differentiate into M2-like macrophages, which have a high production of transforming growth factor-β (TGF-β) and interleukin (IL)-10. The analysis of bioinformatics indicated that exosomal miR-21-5p is closely related to TAM differentiation and is associated with unfavorable prognosis in HCC. Overexpressing miR-21-5p in human monocyte-derived leukemia (THP-1) cells induced down-regulation of IL-1β levels; however, it enhanced production of IL-10 and promoted the malignant growth of HCC cells in vitro. A reporter assay confirmed that miR-21-5p directly targeted Ras homolog family member B (RhoB) 3'-untranslatedregion (UTR) in THP-1 cells. Downregulated RhoB levels in THP-1 cells would weaken mitogen-activated protein kinase (MAPK) axis signaling pathways. Taken together, tumor-derived miR-21-5p promote the malignant advance of HCC, which mediated intercellular crosstalk between tumor cells and macrophages. Targeting M2-like TAMs and intercepting their associated signaling pathways would provide potentially specific and novel therapeutic approaches for HCC treatment.

Keywords: HCC-derived exosomes; RhoB; macrophages; miR-21-5p; polarization.

Figure 1

M2-like TAMs are associated with an unfavorable prognosis of HCC. (A,B) UMAP plot showing the expression levels of characteristic genes (CD68, CD5L, and MRC1) in macrophages in tumor and normal tissues. Violin plots indicating the expression of selected genes in macrophages from HCC samples. Data are from the online tool accessed at: “http://cancer-pku.cn:3838/HCC/ (accessed on 25 August 2022)”. (C) IHC double staining characterizes the distribution of macrophages in HCC, and Kaplan–Merier survival curves present the relationship between infiltration of TAM and prognosis of patients. The data are from The Human Protein Atlas “https://www.proteinatlas.org/ (accessed on 25 August 2022)”.

Figure 2

HCC-derived exosomes induced macrophage M2-like polarization. (A) Exosomes (HepG2 and Huh7) derived from HCC cell lines were collected by an ultrafiltration concentration and ultracentrifugation method. The morphology of exosomes was observed by transmission electron microscopy. The exosomes were marked with arrows. Scare bar: 100 nm. (B) Western blot analysis was used to detect exosome biomarkers (HepG2 and Huh7) derived from HCC cell lines, including Hsp70 and TSG101. (C) The sizes of HepG2 cell-derived exosomes were detected using a Mastersizer 3000. (D) Morphology characteristics of THP-1 cells after PMA treatment. THP-1 cells were seeded in 6-wells by 1 × 10⁵ cells per well and treated with PMA for 48 h. (E) qPCR was used to detect CD68 levels in THP-1 cells after PMA treatment, *** p < 0.001. (F) Cytokines were detected by qPCR after HepG2-derived exosomes treated macrophages for 48 h. ** p < 0.01, * p < 0.05.

Figure 3

HCC-derived exosomes contain large amounts of miR-21-5p. (A) Heatmap shows top 30 miRNA (in the red box) in HCC cell line-derived exosomes (GSE106452). (B) Volcano plot shows differentially expressed miRNAs in TCGA LIHC dataset. (C) Venn diagram shows the intersection of up-regulated miRNA in GES106452 and TCGA LIHC datasets. (D) Violin plots show the expression of mir-21-5p in different types of tissue in TCGA LIHC. (E) Overall survival curves of TCGA LIHC data indicate the relationship between miRNA expression level and patient prognosis.

Figure 4

Tumor-derived exosomes regulate macrophage M2 polarization. (A) HCC cell line-derived exosomes were labeled by PKH67 and co-cultured with THP-1 cells for 24 h. The results were observed by fluorescence microscopy. (B) miR-21-5p level was detected by qPCR. (C) Transcriptional level of cytokines of THP-1 cells after mir-21-5p mimics transfection. 1 × 10⁵ THP-1 cells were seeded in 6-well plates and were treated with PMA for 48 h. miR-21-5p mimics were transfected into THP-1 cells using lipofectamine 2000. RNA was extracted from cells to perform qPCR detection. (D) The supernatant of THP-1 was used to treat HCC tumor cells (HepG2, Hep3B, and Huh7) for 48 h; cell viability was detected using CKK-8 assay. *** p < 0.001.

Figure 5

RhoB is a potential target for miR-21-5p. (A) Venn diagram shows the predicted targets of miR-21-5p, which were made by two kinds of bioinformatic tools. (B) Heatmap showing the DEGs between M1- and M2-polarized macrophages. Data were from GES 66805 and GES 95405. (C) Enrichment analysis of integrated DEGs. The color represents the p-value of the terms, while the x-axis represents different gene categories. (D) The network was constructed with the target genes of miR-21-5p and DEGs in macrophage polarization using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Blue boxes are the predicted targets of mir-21-5p, and green boxes are up-regulated genes, while the orange boxes are down-regulated genes during macrophage M2 polarization.

Figure 6

RhoB is the direct target for miR-21-5p in polarization of macrophages. (A) Sequence diagrams showing wild type and mutant type RhoB 3′UTR for miR-21-5p. (B) We transfected miR-21-5p mimics into THP-1 cells using lipofectamine 2000 and detected RhoB level using Western-blot analysis. (C) Reporter assay: THP-1 cells were transfected with wild type or mutant type RhoB 3′UTR vectors as indicated. After 24 h, the cells were infected with miRNA mimics. 14 h later, luciferase activity of reporters was measured, ** p < 0.05. (D) We transfected RhoB siRNA into THP-1 cells, and detected protein levels using Western-blot analysis as indicated.

Figure 7

HCC-derived exosomal miR-21-5p regulated macrophage polarization by target RhoB. (A) THP-1 cells were seeded on upper layer after transfection with miRNA sponges. HCC cells were seeded on lower layer. Two kinds of cells were cultured for 48 h. (B) The levels of targets of miR-21-5p in HCC cells were detected using Western-blot analysis. (C) Cytokines in THP-1 were detected by qPCR, * p < 0.05.