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. 2014 Jun 28;106(8):dju162.
doi: 10.1093/jnci/dju162. Print 2014 Aug.

Effect of miR-142-3p on the M2 macrophage and therapeutic efficacy against murine glioblastoma

Shuo Xu  1 , Jun Wei  1 , Fei Wang  1 , Ling-Yuan Kong  1 , Xiao-Yang Ling  1 , Edjah Nduom  1 , Konrad Gabrusiewicz  1 , Tiffany Doucette  1 , Yuhui Yang  1 , Nasser K Yaghi  1 , Virginia Fajt  1 , Jonathan M Levine  1 , Wei Qiao  1 , Xin-Gang Li  1 , Frederick F Lang  1 , Ganesh Rao  1 , Gregory N Fuller  1 , George A Calin  1 , Amy B Heimberger  2
Affiliations

Effect of miR-142-3p on the M2 macrophage and therapeutic efficacy against murine glioblastoma

Shuo Xu et al. J Natl Cancer Inst. .

Abstract

Background: The immune therapeutic potential of microRNAs (miRNAs) in the context of tumor-mediated immune suppression has not been previously described for monocyte-derived glioma-associated macrophages, which are the largest infiltrating immune cell population in glioblastomas and facilitate gliomagenesis.

Methods: An miRNA microarray was used to compare expression profiles between human glioblastoma-infiltrating macrophages and matched peripheral monocytes. The effects of miR-142-3p on phenotype and function of proinflammatory M1 and immunosuppressive M2 macrophages were determined. The therapeutic effect of miR-142-3p was ascertained in immune-competent C57BL/6J mice harboring intracerebral GL261 gliomas and in genetically engineered Ntv-a mice bearing high-grade gliomas. Student t test was used to evaluate the differences between ex vivo datasets. Survival was analyzed with the log-rank test and tumor sizes with linear mixed models and F test. All statistical tests were two-sided.

Results: miR-142-3p was the most downregulated miRNA (approximately 4.95-fold) in glioblastoma-infiltrating macrophages. M2 macrophages had lower miR-142-3p expression relative to M1 macrophages (P = .03). Overexpression of miR-142-3p in M2 macrophages induced selective modulation of transforming growth factor beta receptor 1, which led to subsequent preferential apoptosis in the M2 subset (P = .01). In vivo miR-142-3p administration resulted in glioma growth inhibition (P = .03, n = 5) and extended median survival (miR-142-3p-treated C57BL/6J mice vs scramble control: 31 days vs 23.5 days, P = .03, n = 10; miR-142-3p treated Ntv-a mice vs scramble control: 32 days vs 24 days, P = .03, n = 9), with an associated decrease in infiltrating macrophages (R (2) = .303).

Conclusions: These data indicate a unique role of miR-142-3p in glioma immunity by modulating M2 macrophages through the transforming growth factor beta signaling pathway.

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Figures

Figure 1.
Figure 1.
miR-142-3p expression in gliomas. A) Heatmaps demonstrating the microRNA (miRNA) expression pattern in glioblastoma-infiltrating macrophages compared with matched peripheral blood monocytes (n = 4) using the Human miRNA OneArray Microarray v2. With a mean 4.95-fold decrease in level relative to matched peripheral blood monocytes, miR-142-3p emerged as a leading downregulated candidate. B) Total RNA was extracted from a validating set of glioma cancer stem cells (gCSCs; round dots; n = 5), glioma cell lines (cubes; n = 2), glioblastoma tumor tissues (triangles; n = 4), healthy donor peripheral blood CD14+ monocytes (inverted triangles; n = 3), glioblastoma patient peripheral blood CD14+ monocytes (diamonds; n = 6), and glioblastoma infiltrating CD11b+ macrophages (empty circles; n = 3). Analysis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated the various miR-142-3p expressions among different samples. Of note, miR-142-3p is downregulated within the glioblastoma-infiltrating macrophages relative to peripheral blood monocytes. An unpaired t test was used to calculate the two-sided P values. Central horizontal lines are means, and error bars represent standard deviations. *P < .001.
Figure 2.
Figure 2.
Preferential expression of miR-142-3p in M1 macrophages. Human CD14+ monocytes were incubated with granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) to induce M1 and M2 macrophages, respectively. A) The M1 and M2 macrophages have a distinctive ex vivo morphology. Scale bar = 20 µM. B) Cell surface expression of CD14, CD45, CD11b, CD80, CD86, major histocompatibility complex II (MHC II), CD68, and CD163 were evaluated by flow cytometry. Representative results are shown. Similar results were observed in 3 replicates. The gray histogram denotes M1 macrophages, black denotes M2 macrophages, and the dotted line represents the isotype control. C) The phagocytic activities of M1 and M2 macrophages were measured by fluorescent uptake and summarized. Error bars represent standard deviations. A paired t test was used to calculate the two-sided P values. n = 6. ***P < .001. D) The miR-142-3p expression was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Its expression was downregulated during macrophage differentiation but preferentially in the immunosuppressive M2 macrophages relative to the proinflammatory M1 macrophages. Error bars represent standard deviations. A paired t test was used to calculate the two-sided P values. n = 6. *P = .03.
Figure 3.
Figure 3.
miR-142-3p interacts with the transforming growth factor beta receptor 1 (TGFBR1) pathway. A) Western blot analyses of the predicted miR-142-3p targets indicated by bioinformatics tools in M1 and M2 macrophages untreated (blank), transfected with scramble control (ctrl), transfected with miR-142-3p (miR), or transfected with anti-miR-142-3p (anti). The predicted targets include integrin beta 8 (ITGB8), integrin alpha V (ITGAV), transforming growth factor beta 2 (TGFB2), SMAD4, NF-κB, and TGFBR1. Similar results were observed in 3 replicates. B) miR-142-3p overexpression inhibits the downstream target, p-SMAD2, in M2 macrophages. The M1 and M2 macrophages were treated with transforming growth factor-β1 (TGF-β1), transfected with scramble control (ctrl) or miR-142-3p (miR), and then measured for p-SMAD2. Similar results were observed in 3 replicates. C) Sequences of the predicted miR-142-3p binding site on the wild-type TGFBR1 3’-untranslated region (3’-UTR; upper) and the mutated TGFBR1 3’-UTR sequence (lower), which potentially disrupts miR-142-3p binding (middle). D) The relative luciferase activity in HeLa cells after transfection with miR-142-3p in conjunction with the parental luciferase vector (Vector), the wild-type (WT) TGFBR1 3’-UTR, or the miR-142-3p binding site mutant reporter construct (Mut). Luciferase activity is shown relative to the parental luciferase vector, and error bars represent standard deviations. Similar results were observed in 3 replicates.
Figure 4.
Figure 4.
miR-142-3p transfection and transforming growth factor beta receptor 1 (TGFBR1) blockade induces selective apoptosis in M2 macrophages. A) Representative flow cytometry analyses and summarized data demonstrating more early apoptosis (Annexin V+7-AAD-) and late apoptosis/cell death (Annexin V+7-AAD+) induced in M2- (empty circles) than in M1- (round dots) committed macrophages at 48 hours after the scramble control (ctrl) or miR-142-3p (miR) transfection. Error bars represent standard deviations. After excluding for outliers detected by the Grubbs test, a paired t test was used to calculate the two-sided P values. n = 8. *P = .01. B) Representative flow cytometry analyses and summarized dataset of blockade of TGFBR1 by the antagonists SB431542 (left two columns; n = 6) and LY364947 (right two columns; n = 6) demonstrating induced preferential M2 macrophage apoptosis. The medium dimethyl sulfoxide (DMSO) was examined as control. Round dots are M1-committed monocytes, and empty circles are M2-committed monocytes. A paired t test was used to calculate the two-sided P values. Error bars represent standard deviations. *P = .04 for SB431542; **P = .001 for LY364947.
Figure 5.
Figure 5.
miR-142-3p inhibits in vivo glioma growth. The local treatment schema (A) and volume of subcutaneous (s.c.) GL261 tumors (B) in C57BL/6J mice treated with scramble control (ctrl) and miR-142-3p (miR) starting on day 5 (n = 5 per group). Empty circles are scramble controls, and triangles are miR-142-3p–treated mice. Standard deviations are shown. Linear mixed models were fit to assess tumor growth, and two-sided F test was used. *P = .03. The in vivo experiment was duplicated with similar results. The intravenous (i.v.) treatment schema (C) and graph of the Kaplan–Meier estimate of survival time in C57BL/6J mice implanted with intracerebral (i.c.) GL261 gliomas (D), which showed that miR-142-3p improved survival in the miR-142-3p-treated group (miR; round dots) relative to the scramble controls (ctrl; empty triangles). Log-rank test was used to compare overall survival between groups. n = 10 per group. *P = .03.
Figure 6.
Figure 6.
miR-142-3p inhibits glioma-infiltrating macrophages. The intravenous (i.v.) treatment schema (A) and graph of the Kaplan–Meier estimate of survival time (B) demonstrating improved survival in miR-142-3p–treated (miR) Ntv-a mice transfected with the intracerabral (i.c.) RCAS-platelet-derived growth factor B (PDGFB) + RCAS-B-cell lymphoma 2 (Bcl-2) transgenes compared with scramble control (ctrl). Empty triangles are scramble controls, and round dots are miR-142-3p–treated mice. Log-rank test was used to compare overall survival between groups. n = 9 per group. *P = .03. C) Immunohistochemistry demonstrating staining with anti-F4/80 antibodies to identify glioma-infiltrating macrophages. Left panel: representative images of mice treated with scramble control (ctrl) and miR-142-3p (miR), respectively. Scale bar = 50 µm. Right panel: quantification of glioma-infiltrating macrophage and comparison between the two groups. Triangles are scramble controls, and round dots are miR-142-3p–treated mice. A paired t test was used to calculate the two-sided P values. n = 8 per group. *P = .009. D) The correlation between the percentage of glioma-infiltrating F4/80+ macrophages and the survival duration of miR-142-3p treated mice. n = 8. R 2 = .303.
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References

    1. Gabriely G, Wurdinger T, Kesari S, et al. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol. 2008;28(17):5369–5380. - PMC - PubMed
    1. Iliopoulos D, Jaeger SA, Hirsch HA, Bulyk ML, Struhl K. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer. Mol Cell. 2010;39(4):493–506. - PMC - PubMed
    1. Andreopoulos B, Anastassiou D. Integrated analysis reveals hsa-miR-142 as a representative of a lymphocyte-specific gene expression and methylation signature. Cancer Inform. 2012;11:61–75. - PMC - PubMed
    1. Chan E, Patel R, Nallur S, et al. MicroRNA signatures differentiate melanoma subtypes. Cell Cycle. 2011;10(11):1845–1852. - PMC - PubMed
    1. Wang F, Wang XS, Yang GH, et al. miR-29a and miR-142-3p downregulation and diagnostic implication in human acute myeloid leukemia. Mol Biol Rep. 2012;39(3):2713–2722. - PubMed

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