Epstein-Barr virus encoded miR-BART11 promotes inflammation-induced carcinogenesis by targeting FOXP1

Abstract

Epstein-Barr virus (EBV) infection and chronic inflammation are closely associated with the development and progression of nasopharyngeal carcinoma (NPC) and gastric cancer (GC), and the infiltration of inflammatory cells, including tumor-associated macrophages (TAMs), is often observed in these cancers. EBV encodes 44 mature micro RNAs (miRNAs), but the roles of only a few EBV-encoded miRNA targets are known in cancer development, and here, our aim was to elucidate the effects of EBV-miR-BART11 on FOXP1 expression, and potential involvement in inflammation-induced carcinogenesis. We constructed an EBV miRNA-dependent gene regulatory network and predicted that EBV-miR-BART11 is able to target forkhead box P1 (FOXP1), a key molecule involved in monocyte to macrophage differentiation. Here, using luciferase reporter assay, we confirmed that EBV-miR-BART11 directly targets the 3'-untranslated region of FOXP1 gene, inhibits FOXP1 induction of TAM differentiation, and the secretion of inflammatory cytokines into the tumor microenvironment, inducing the proliferation of NPC and GC cells. FOXP1 overexpression hindered monocyte differentiation and inhibited NPC and GC cells growth. Our results demonstrated that EBV-miR-BART11 plays a crucial role in the promotion of inflammation-induced NPC and GC carcinogenesis by inhibiting FOXP1 tumor-suppressive effects. We showed a novel EBV-dependent mechanism that may induce the carcinogenesis of NPC and GC, which may help define new potential biomarkers and targets for NPC and GC diagnosis and treatment.

Keywords: EBV-miR-BART11; Epstein-Barr virus; FOXP1; gastric cancer; nasopharyngeal carcinoma.

Figure 1. FOXP1 is a direct target of EBV-miR-BART11

A. Three binding sites of EBV-miR-BART11-3p and EBV-miR-BART11-5p were predicted in the FOXP1 3′-UTR, including 3082 bp-3106 bp (I), 3917 bp-3998 bp (II), and 5943 bp-5970 bp (III). Wild-type (FOXP1-WT) and mutant (FOXP1-mutant) sequences were used to validate these predictions. B. The expression of exogenous EBV-miR-BART11-3p (left) and EBV-miR-BART11-5p (right) was detected by qRT-PCR. FOXP1 C. mRNA and D. protein expression levels in 5-8F, HK-1, and AGS cells after EBV-miR-BART11 treatment. β-actin served as loading control. E. Luciferase reporter assay, using reporter vectors containing either wild-type (FOXP1-WT) or mutant (FOXP1-mutant) FOXP1 3′-UTR, and EBV-miR-BART11 or non-targeting control, was performed in order to identify the direct binding of EBV-miR-BART11 to the FOXP1 3′-UTR in 5-8F cells. F. EBV-miR-BART11 and FOXP1 expression levels in NPC and control specimens were detected by qRT-PCR. N, non-tumor nasopharyngeal epithelium (n = 10); T, NPC (n = 30). Representative images or data expressed as mean ± SD of the measurements obtained in three separate experiments are presented (ND: not detected; *p < 0.05; **p < 0.01;***p < 0.001).

Figure 1. FOXP1 is a direct target of EBV-miR-BART11

A. Three binding sites of EBV-miR-BART11-3p and EBV-miR-BART11-5p were predicted in the FOXP1 3′-UTR, including 3082 bp-3106 bp (I), 3917 bp-3998 bp (II), and 5943 bp-5970 bp (III). Wild-type (FOXP1-WT) and mutant (FOXP1-mutant) sequences were used to validate these predictions. B. The expression of exogenous EBV-miR-BART11-3p (left) and EBV-miR-BART11-5p (right) was detected by qRT-PCR. FOXP1 C. mRNA and D. protein expression levels in 5-8F, HK-1, and AGS cells after EBV-miR-BART11 treatment. β-actin served as loading control. E. Luciferase reporter assay, using reporter vectors containing either wild-type (FOXP1-WT) or mutant (FOXP1-mutant) FOXP1 3′-UTR, and EBV-miR-BART11 or non-targeting control, was performed in order to identify the direct binding of EBV-miR-BART11 to the FOXP1 3′-UTR in 5-8F cells. F. EBV-miR-BART11 and FOXP1 expression levels in NPC and control specimens were detected by qRT-PCR. N, non-tumor nasopharyngeal epithelium (n = 10); T, NPC (n = 30). Representative images or data expressed as mean ± SD of the measurements obtained in three separate experiments are presented (ND: not detected; *p < 0.05; **p < 0.01;***p < 0.001).

Figure 2. EBV-miR-BART11 promotes monocyte differentiation of THP-1 cells by attenuating FOXP1 expression

A. FOXP1 expression at mRNA (left) and protein (right) levels, during the PMA-induced differentiation of monocytic THP-1 cells. B. The expression of EBV-miR-BART11 (left) and FOXP1 (middle, mRNA; right, protein) was examined by qRT-PCR and western blotting, respectively, in THP-1 cells infected with lentivirus encoding EBV-miR-BART11. C. The effects of EBV-miR-BART11 and FOXP1 on monocyte differentiation. Morphological changes were monitored in PMA-induced THP-1 cells following FOXP1 overexpression vector or EBV-miR-BART11 precursor vector transfection. THP-1 cell differentiation was determined by the viability of adherent cells, using MTT assay. Data represent mean ± SD of OD values obtained in three separate experiments (ND: not detected; **p < 0.01; ***p < 0.001).

Figure 3. EBV-miR-BART11-transfected macrophages are hyperresponsive to LPS

A. Pro-inflammatory cytokine (IL-1β, IL-6, and IL-8) expression in PMA-treated THP-1 cells. B. Differentiated THP-1 (D-THP-1) cells, stimulated with LPS in the presence of exogenous FOXP1 overexpression (upper panel) or EBV-miR-BART11 (lower panel). Data are expressed as mean ± SD of the results obtained from each group of cells in three separate experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4. Effect of FOXP1 and EBV-miR-BART11 expression on the local inflammatory response in epithelial cells

The expression of inflammatory factors (IL-1β, IL-6, and IL-8), measured in 5-8F and HK-1 NPC and AGS GC epithelial cells transfected with FOXP1 overexpression vector A. or EBV-miR-BART11 precursor vector B. and treated with LPS or conditioned media collected from the untreated differentiated-THP-1 cells (D-THP) or LPS-treated D-THP-1 cells (D-THP-LPS). Data are represented as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. Effect of EBV-miR-BART11 and FOXP1 on cancer cell proliferation

The effect of FOXP1 or EBV-miR-BART11 overexpression, or FOXP1 siRNA knockdown (siFOXP1) on cell proliferation was investigated by the A. MTT assay and B. colony formation assay in 5-8F, HK-1, and AGS cells. C. Cell cycle analysis of cell cycle progression by flow cytometric analysis. All data were normalized to the vector-only control cell proliferation rate, and the results are expressed as mean ± SD of the results obtained in three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. Effect of EBV-miR-BART11 and FOXP1 on cancer cell proliferation

The effect of FOXP1 or EBV-miR-BART11 overexpression, or FOXP1 siRNA knockdown (siFOXP1) on cell proliferation was investigated by the A. MTT assay and B. colony formation assay in 5-8F, HK-1, and AGS cells. C. Cell cycle analysis of cell cycle progression by flow cytometric analysis. All data were normalized to the vector-only control cell proliferation rate, and the results are expressed as mean ± SD of the results obtained in three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6. Effect of EBV-miR-BART11 and FOXP1 on inflammation-induced cancer cell proliferation

The proliferation of 5-8F, HK-1, and AGS cells transfected with EBV-miR-BART11 precursor vector A. FOXP1 siRNA B. or FOXP1 overexpression vector C. cultured in the conditioned media collected from LPS-treated differentiated THP-1 cells (D-THP-LPS). Data are expressed as mean ± SD of cell growth following different treatments, of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7. EBV-miR-BART11-mediated NF-κB activation via FOXP1 inhibition

A. The expression of FOXP1 and NF-κB in 5-8F, HK-1, and AGS epithelial cancer cells and THP-1 monocytes, transfected with FOXP1, EBV-miR-BART11, or siFOXP1, and determined by western blotting and densitometry. B. NF-κB transcriptional activity, assessed by luciferase reporter assay in 5-8F cells transfected with the FOXP1 overexpression vector, EBV-miR-BART11 precursor vector, or siFOXP1. Data shown are representative images or expressed as mean ± SD of different groups of cells from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.