The mechanism of lncRNA-CRNDE in regulating tumour-associated macrophage M2 polarization and promoting tumour angiogenesis

Abstract

M2 macrophages can promote liver cancer metastasis by promoting tumour angiogenesis; however, the mechanism underlying macrophage polarization has not been completely revealed. In this study, we mainly explored the mechanism underlying long non-coding RNA-CRNDE (lncRNA-CRNDE) in regulating M2 macrophage polarization and promoting liver cancer angiogenesis. The expression of CRNDE was up-regulated or down-regulated in THP-1 cells (CRNDE^-/- -THP-1 cells and pcDNA3.1-CRNDE-THP-1). THP-1 cells were co-cultured with liver cancer cell line H22, and M2 polarization was induced in THP-1 by IL-4/13 to simulate tumour-induced macrophage polarization. As a result, after CRNDE overexpression, THP-1 cell viability was up-regulated, the expression of M2 membrane marker CD163 was up-regulated, and the proportion of F4/80 + CD163+ cells was also up-regulated. ELISA assay showed that the expression of M2 markers (including TGF-β1 and IL-10) and chemokines (including CCl22 and CCL22) was up-regulated, and the expression of key signals (including STAT6, JAK-1, p-AKT1, and Arg-1) was also up-regulated, which were significantly different compared with the control group (Con). In addition, the intervention effect of CRNDE on THP-1 was consistent between co-culture with H22 cells and IL-4/13 induction assay. The induced M2 THP-1 cells were co-cultured with HUVEC. As a result, THP-1 cells with CRNDE overexpression can promote the migration and angiogenesis of HUVEC cells in vitro and simultaneously up-regulate the expression of Notch1, Dll4 and VEGFR2, indicating that THP-1 M2 polarization induced by CRNDE could further promote angiogenesis. The H22 cell tumour-bearing mouse model was constructed, followed by injection of CRNDE anti-oligosense nucleotides and overexpression plasmids to interfere CRNDE expression in tumour-bearing tissues. Consequently, down-regulation of CRNDE could down-regulate tumour volume, simultaneously down-regulate the expression of CD163 and CD31 in tissues, decrease the expression of key proteins (including JAK-1, STAT-6, p-STAT6 and p-AKT1), and down-regulate the expression of key angiogenesis-related proteins (including VEGF, Notch1, Dll4 and VEGFR2). In this study, we found that CENDE could indirectly regulate tumour angiogenesis by promoting M2 polarization of macrophages, which is also one of the mechanisms of microenvironmental immune regulation in liver cancer.

Keywords: M2 polarization; long non-coding RNA-CRNDE; macrophages; microenvironment; tumour blood vessels.

FIGURE 1

Effects of CRNDE on M2 polarization of THP‐1 cells. A, Cell viability results (n = 5): The cell viability of the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group, while the cell viability of the THP‐1‐shRNA group was significantly lower than that of the THP‐1‐Con group. The expression of CRNDE was associated with the viability of THP‐1 cells. Comparison with THP‐1‐Con, *P <.05; **P <.01. B and C, The proportion of F4/80 + CD163+M2 macrophages by flow cytometry (n = 5): The proportion of F4/80 + CD163+M2 macrophages in the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group, while the proportion of F4/80 + CD163+M2 macrophages in the THP‐1‐shRNA group was significantly lower than that of the THP‐1‐Con group. Comparison between groups, *P <.05; **P <.01. D, CD163 expression by immunofluorescence staining (n = 3): The expression of CD163 in the THP‐1‐pcDNA3.1 group was significantly higher than that in the THP‐1‐Con group, while the expression of CD163 in the THP‐1‐shRNA group was lower than that of the THP‐1‐Con group

FIGURE 2

Effects of CRNDE on M2 polarization of THP‐1 cells and the mechanism. A‐E, Detection of M2 macrophage marker expression by CRNDE (n = 5): The expression of M2 macrophage markers in the THP‐1‐pcDNA3.1 group was significantly increased, which was significantly different from the THP‐1‐Con group. Meanwhile, the expression of M2 macrophage markers in THP‐1‐shRNA group was lower than that of the THP‐1‐Con group. Comparison between groups, *P <.05; **P <.01. F‐I, Effects of CRNDE on JAK‐STAT6 and AKT1 signal expression (n = 3): The expression of JAK1, STAT6, p‐STAT6, AKT1 and p‐AKT1 in the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group. Meanwhile, the expression level in the THP‐1‐shRNA group was lower than that of the THP‐1‐Con group. Comparison between groups, *P <.05; **P <.01

FIGURE 3

Effects of CRNDE on M2 polarization of THP‐1 induced by liver cancer cells H22. A, Cell viability results (n = 5): The cell viability of the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group. The expression of CRNDE was associated with THP‐1 cell viability. Comparison with THP‐1‐Con, *P <.05; **P <.01. B and C, Proportion of F4/80 + CD163+M2 macrophages by flow cytometry (n = 5): The proportion of F4/80 + CD163+M2 macrophages in the THP‐1‐pcDNA3.1 cells was significantly higher than that of THP‐1‐Con group, while the proportion in the THP‐1‐shRNA group was significantly lower than that of THP‐1‐Con group. Comparison between groups, *P <.05; **P <.01. D, CD163 expression by immunofluorescence staining (n = 3): The expression of CD163 in the THP‐1‐pcDNA3.1 group was significantly higher than that in the THP‐1‐Con group, while the expression of CD163 in the THP‐1‐shRNA group was lower than that of the THP‐1‐Con group

FIGURE 4

Effects of CENDE on the expression of M2 macrophage markers and key protein expression. A‐E, Detection of M2 macrophage marker expression (n = 5): The expression of M2 macrophage markers was significantly increased in the THP‐1‐pcDNA3.1 group, which was significantly different from the THP‐1‐Con group. Meanwhile, the expression in the THP‐1‐shRNA group was lower than that of THP‐1‐Con group. Comparison between groups, *P <.05; **P <.01. F‐I, Effects of CRNDE on JAK‐STAT6 and AKT1 signal expression (n = 3): The expression of JAK1, STAT6, p‐STAT6, AKT1 and p‐AKT1 in the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group. Meanwhile, the expression level in the THP‐1‐shRNA group was lower than that of the THP‐1‐Con group. Comparison between groups, *P <.05; **P <.01

FIGURE 5

Effects of IL4/13‐induced M2 macrophages on in vitro angiogenesis. A, Effects of M2 macrophages on HUVEC cell viability (n = 5): The cell viability of THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group. CRNDE expression was associated with THP‐1 cell viability. Comparison with THP‐1‐Con group, *P <.05; **P <.01. B, Results of HUVEC cell migration ability (n = 5): The cell migration ability of the THP‐1‐pcDNA3.1 group was significantly increased than that of the THP‐1‐Con group. C, In vitro tube formation assay of HUVEC and chick embryo chorioallantoic membrane assay (n = 5): the vessel formation of the THP‐1‐pcDNA3.1 group was significantly increased than that of THP‐1‐Con group. D and E, Expression of key signal proteins of angiogenesis (n = 3): The expression of Notch1, Dll4 and VEGFR2 proteins in the THP‐1‐pcDNA3.1 group was significantly higher than that in the THP‐1‐Con group, and the expression in the THP‐1‐shRNA group was lower than the THP‐1‐Con group. Comparison with THP‐1‐Con, *P <.05; **P <.01

FIGURE 6

H22‐induced M2 macrophages‐induced angiogenesis. A, HUVEC cell viability results (n = 5): The cell viability of the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con. Comparison with THP‐1‐Con, *P <.05; **P <.01. B, HUVEC cell migration assay (n = 5): The cell migration capacity of THP‐1‐pcDNA3.1 group was significantly enhanced than that of THP‐1‐Con group. C, In vitro tube formation assay of HUVEC and chick embryo chorioallantoic membrane assay (n = 5): The vessel formation ability of THP‐1‐pcDNA3.1 group was significantly enhanced than THP‐1‐Con group. D and E, Expression of key signal proteins for angiogenesis (n = 3): The protein expression of Notch1, Dll4 and VEGFR2 in the THP‐1‐pcDNA3.1 group was significantly higher than that of the THP‐1‐Con group, and the expression in the HP‐1‐shRNA group was lower than that of the THP‐1‐Con group. Comparison with THP‐1‐Con, *P <.05; **P <.01

FIGURE 7

Effects of CRNDE on M2 macrophages and tumour angiogenesis in tumour‐bearing mice. A, Immunohistochemical staining of CD163 and CD31 and H&E staining results of mouse tumour tissues (n = 5): H&E staining showed no obvious tissue lesions among the three groups of mice. The level of CD163 and CD31 in the anti‐CRNDE group was lower than Con group, while the expression of CD163 and CD31 was higher in the pcDNA3.1‐CRNDE group than the Con group. B and C, Expression of key protein of M2 macrophage polarization (n = 3): The protein expression in the pcDNA3.1‐CRNDE group was significantly higher than that in the Con group, while the expression of the anti‐CRNDE group was lower than that of the Con group. Comparison with Con, * P <.05; **P <.01. D and E, Expression of key proteins in angiogenesis (n = 3): The protein expression in the pcDNA3.1‐CRNDE group was significantly higher than that in the Con group, while the expression of the anti‐CRNDE group was lower than that of the Con group. Comparison with Con, *P <.05; **P <.01