MiR27a Promotes the Development of Macrophage-like Characteristics in 3T3-L1 Preadipocytes

Abstract

Recruitment and polarization of classically activated (M1) macrophages within adipose tissue contribute to chronic low-grade inflammation in obesity. Adipose tissue precursor cells exhibit the capacity to develop macrophage-like characteristics and adipocyte-derived miR27a is known to promote reprogramming of somatic cells. It was unknown whether exogenous addition of miR27a promote the development of macrophage-like characteristics of adipose precursor cells. We examined macrophage surface antigen, phagocytosis and migration ability in 3T3-L1 preadipocytes transfected with miR27a mimics. Transfection of 3T3-L1 preadipocytes with miR27a mimics increased phagocytosis and migration and increased the number of cells expressing the macrophage makers F4/80 and MHC compared to controls. M2 and CD206 macrophage markers were unaltered. In addition, transfection of 3T3-L1 preadipocytes with miR27a mimics reduced PPARγ expression, activated NF-κB and promoted secretion of the inflammatory cytokines MCP-1, TNF-α and IL-1β compared to controls. The level of anti-inflammatory factors Arg-1, IL-10, Ym1 and Fizz1 were unaltered. Secretion of miR27a was increased in conditioned medium prepared from palmitic acid-treated differentiated 3T3-L1 adipocytes compared to controls. Incubation of 3T3-L1 preadipocytes with this conditioned medium increased phagocytosis and migration compared to controls. Finally, conditioned medium prepared from differentiated 3T3-L1 adipocytes transfection with miR27a inhibitors reduced phagocytosis and migration in 3T3-L1 preadipocytes compared to controls. The data indicate that PPARγ agonists may reverse the activation of NF-κB pathway mediated by miR27a overexpression and reduce phagocytosis and migration of adipose precursor cells. In addition, miR27a may promote the development of macrophage-like characteristics in 3T3-L1 preadipocytes.

Keywords: 3T3 cells; inflammation; macrophage-like; miR27a; obesity; preadipocytes.

Figure 1

miR27a promotes phagocytosis in 3T3-L1 preadipocytes. A. Transfection efficiency of miR27a in control (con) and miR27a mimic (miR27a(+)) incubated cells. Red fluorescence was the miR-27a transfected in cells. B. Relative expression of miR27a in control (con) and miR27a mimic (miR27a(+)) incubated cells. Data are shown as the mean ±SEM, n=6, ***p<0.001 compared to control. C. Phagocytic activity in control (con) and cells transfected with miR27a mimic for up to 72 h. n=5, ***p<0.001 compared to control. D. and E. Phagocytic activity in control (con) and cells transfected with miR27a mimic (miR27a(+)) using flow cytometry. n=3, **p<0.01, compared to control.

Figure 2

miR27a influences expression of the macrophage cell surface marker. A. Cell surface F4/80 expression in control (A04 con) and miR27a mimic (A07 27a overexpress) incubated cells. Negative control (A01 negative). B. Relative F4/80 level in control (con) and miR27a mimic (miR27a(+)) incubated cells. C. Cell surface MHC expression in control (B05 con) and miR27a mimic (B02 27a overexpress) incubated cells. Negative control (A01 negative). D. Relative MHC level in control (con) and miR27a mimic (miR27a(+)) incubated cells. E. Cell surface CD206 expression in control (B07 con) and miR27a mimic (C07 27a overexpress) incubated cells. Negative control (A02 negative). F. Relative CD206 level in control (con) and miR27a mimic (miR27a(+)) incubated cells.

Figure 3

Transfection of 3T3-L1 preadipocyte cells with miR27a mimics increases the number of F4/80 positive cells expressing MHC. A. Cell surface MHC expression in control (B07 con) and miR27a mimic (B02 miR27a overexpressing) incubated cells. Negative control (A01 negative). B. Relative MHC level of F4/80 positive cells in control (con) and miR27a mimic (miR27a(+)) incubated cells. C. Cell surface CD206 expression in control (B07 con) and miR27a mimic (C07 miR27a overexpress) incubated cells. Negative control (A02 negative). D. Relative CD206 level of F4/80 positive cells in control (con) and miR27a mimic (miR27a(+)) incubated cells. n=3, *p<0.05, compared to control.

Figure 4

miR27a enhances 3T3-L1 preadipocytes cell migration. A. Cells were cultured on Transwell^® plates and incubated in the absence (con) or presence of miR27a mimic (miR27a(+)) for up to 10 h and stained with crystal violet. (100 x magnification). A relative micrograph is depicted. B: The number of migrating cells. n=3, ***p<0.001, compared to control.

Figure 5

miR27a enhances secretion of inflammatory factors and modulates inflammatory signaling in 3T3-L1 preadipocytes. The level of TNFα (A), MCP-1 (B), IL-1 (C), Arg-1 (D), IL-10 (E), Ym1 (F) and Fizz1 (G) in control (con) and miR27a mimic (miR27a(+)) incubated cells. n=6, *p<0.05,**p<0.01, compared to control. H. Western blot analysis of PPARγ, NF-κB, p-NF-κB and IκBα in control (con) and miR27a mimic (miR27a(+)) incubated cells. A representative blot is depicted. I. Relative protein expression of PPARγ, NF-κB, p-NF-κB and IκBα in control (con) and miR27a mimic (miR27a(+)) incubated cells. n=3, *p<0.05,**p<0.01, compared to control.

Figure 6

The PPARγ agonist rosiglitazone inhibits phagocytic and migration ability and inflammatory pathway protein expression in miR27a transfected 3T3-L1 preadipocytes. A. Cells were cultured on Transwell^® plates and incubated in the absence (con) or presence of miR27a mimic (miR27a(+)) or plus or minus 20 μM rosiglitazone after miR27a overexpression for up to 10 h and stained with crystal violet. (100 x magnification). A representative micrograph is depicted. B. The number of migrating cells. Control (con), cells transfected with miR27a mimic or cells transfected with miR27a mimic treated with 20 μM rosiglitazone n=3, ***p<0.001, compared to control. ^###p<0.001, compared to miR27a transfected cells. C. Phagocytic activity in control (con) or cells transfected with miR27a mimic or cells transfected with miR27a mimic plus 20 μM rosiglitazone. n=5, ***p<0.001 compared to control. ^###p<0.001, compared to miR27a transfected cells. D. Western blot analysis of PPARγ, NF-κB, p-NF-κB and IκBα in control (con) and miR27a mimic treated cells (miR27a(+)) incubated plus or minus 20 μM rosiglitazone. A representative blot is depicted. E. Relative protein expression of PPARγ, NF-κB, p-NF-κB and IκBα in control (con) and miR27a mimic (miR27a(+)) treated cells or miR27a mimic treated cells (miR27a(+)) incubated plus 20 μM rosiglitazone. n=3, *p<0.05,**p<0.01, compared to control.. ^##p<0.01, ^###p<0.001, compared to miR27a transfected cells.

Figure 7

miR27a derived from hypertropic adipocytes promotes phagocytic activity and migration of 3T3-L1 preadipocytes. A. Relative level of miR27a in control (con) or CM2 or CM3 medium. n=3, *p<0.05, compared to control. B. Phagocytic activity in 3T3-L1 preadipocytes incubated with control (con) or CM1 or CM2 or CM3 medium. n=3, ***p<0.001, compared to control. C. Cells cultured on transwell plates and incubated in the presence of control (con) or CM1 or CM2 or CM3 medium and then stained with crystal violet. (100 x magnification). A relative micrograph is depicted. D. The number of migrating cells from C. n=3, ***p<0.001, compared to control.