MiR-125b regulates proliferation and apoptosis of nasopharyngeal carcinoma by targeting A20/NF-κB signaling pathway

Abstract

MiR-125b is aberrantly expressed and has a role in the various types of tumors. However, the role and mechanism of miR-125b in nasopharyngeal carcinoma (NPC) are unclear. In this study, we investigated the role and mechanism of miR-125b in NPC. We observed that miR-125b was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa (NNM), and its increment was correlated with poor patient survival, and was an independent predictor for reduced patient survival; miR-125b promoted NPC cell proliferation and inhibited NPC cell apoptosis; in a mouse model, administration of miR-125b antagomir significantly reduced the growth of NPC xenograft tumors. Mechanistically, we confirmed that A20 was a direct target of miR-125b, and found that activation of nuclear factor κB (NF-κB) signaling pathway by A20 mediated miR-125b-promoting NPC cell proliferation and -inhibiting NPC cell apoptosis. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 inhibited NPC cell proliferation, induced NPC cell apoptosis, and reduced the growth of NPC xenograft tumors. Moreover, A20 was significantly downregulated, whereas p-p65(RelA) was significantly upregulated in the NPC tissues relative to normal nasopharyngeal mucosa, and miR-125b level was negatively associated with A20 level, whereas positively associated with p-p65 level. Our data demonstrate that miR-125b regulates NPC cell proliferation and apoptosis by targeting A20/NF-κB signaling pathway, and miR-125b acts as oncogene, whereas A20 functions as tumor suppressor in NPC, highlighting the therapeutic potential of miR-125b/A20/NF-κB signaling axis in the NPC.

Figure 1

The expression of miR-125b, A20, and p-p65 in NPC and association of miR-125b expression levels with the patient survival. (a) QRT-PCR analysis of the expression levels of miR-125b in the 111 NPC tissues and 30 normal nasopharyngeal mucosa (NNM). Three experiments were done; Means, S.D.s, and statistical significance are denoted; ***P<0.001. (b) Kaplan–Meier survival analysis for 111 NPC patients according to the expression levels of miR-125b. NPC patients with high miR-125b expression have a significantly worse disease-free survival (left) and overall survival (right) than those with low miR-125b expression. The log-rank test was used to calculate P-value. (c) A representative result of immunohistochemistry showing the expression of A20 and p-p65 (RelA) in the NNM and NPC tissues. Scale bars=50 μm

Figure 2

The effects of miR-125b on NPC cell proliferation, apoptosis, and xenograft growth. (a) QRT-PCR analysis of miR-125b expression levels in the NPC cell lines CNE2 and CNE2-IR. Analysis of cell proliferation by CCK-8 (b), EdU incorporation (c) and plate clone formation (d) assay in the miR-125b mimic-transfected CNE2 cells, miR-125b inhibitor-transfected CNE2-IR cells and their control cells. (e) Analysis of cell apoptosis by flow cytometry in the miR-125b mimic-transfected CNE2 cells, miR-125b inhibitor-transfected CNE2-IR cells and their control cells. (f) MiR-125b antagomir inhibits in vivo NPC cell growth. The photography of xenograft tumors after 18 days subcutaneous implantation of control or miR-125b antagomir-injected CNE2-IR CNE2 cells (top); growth and weight of the xenograft tumors (bottom). n=5 mice per group. Means, S.D.s, and statistical significance are denoted; *P<0.05; **P<0.01; ***P<0.001; ns, no significance

Figure 3

Target A20 of miR-125b regulates NPC cell proliferation and apoptosis. (a) 3′-UTR dual luciferase reporter assay showing A20 as a direct target of miR-125b in NPC cells. (left) The predicted miR-125b binding sites in the 3′-UTR of wild-type (wt) A20 and mutant (mt) A20 3′-UTR; (middle) Luciferase activity of wt and mt A20 3′-UTR and without A20 3′-UTR dual luciferase reporter vector in the CNE2 cells transfected with control or miR-125b mimic; (right) Western blot analysis showing A20 levels in the miR-125b mimic-transfected CNE2, miR-125 inhibitor-transfected CNE2-IR cells and their respective control cells. (b) Western blot analysis showing A20 levels in the A20 KD CNE2 cells, A20 OE CNE2-IR cells and their respective control cells. (c) Analysis of cell proliferation by CCK-8 (top), EdU incorporation (middle) and plate clone formation (bottom) assay in A20 KD CNE2 cells, A20 OE CNE2-IR cells and their respective control cells. (d) Analysis of cell apoptosis by flow cytometry in the A20 KD CNE2 cells, A20 OE CNE2-IR cells and their respective control cells. Three experiments were done; means, S.D.s, and statistical significance are denoted; **P<0.01; ***P<0.001; ns, no significance. Vector, transfected with an empty vector; KD, knockdown; OE, overexpression

Figure 4

MiR-125b promotes NPC cell proliferation and inhibits NPC cell apoptosis by targeting A20. (a) Analysis of cell proliferation by EdU incorporation (top) and plate clone formation (bottom) assay in the A20 KD CNE2 cells transfected with miR-125b inhibitor and control cells. (b) Analysis of cell apoptosis by flow cytometry in the A20 KD CNE2 cells transfected with miR-125b inhibitor and control cells. (c) Analysis of cell proliferation by EdU incorporation (top) and plate clone formation (bottom) assay in the A20 OE CNE2-IR cells transfected with miR-125b mimic and control cells. (d) Analysis of cell apoptosis by flow cytometry in the A20 OE CNE2-IR cells transfected with miR-125b mimic and control cells. Three experiments were done; Means, S.D.s, and statistical significance are denoted; ***P<0.001; ns, no significance. Vector, transfected with an empty vector; KD, knockdown; OE, overexpression

Figure 5

A20 inhibits in vivo NPC cell growth. (a) The representative photography of xenograft tumors after 18 days subcutaneous implantation of A20 KD CNE2 cells and control cells (top); Growth and weight of xenograft tumors generated by A20 KD CNE2 cells and control cells (bottom). (b) The representative photography of xenograft tumors after 18 days subcutaneous implantation of A20 OE CNE2-IR cells and control cells (top); Growth and weight of xenograft tumors generated by A20 OE CNE2-IR cells and control cells (bottom). (c) Representative results of A20, p-p65 (RelA), TUNEL, and Ki-67 immunohistochemical staining (top) and statistical analysis (bottom) of xenograft tumors generated by A20 KD CNE2 cells, A20 OE CNE2-IR cells and their respective control cells. n=10 mice per group. Original magnification, × 400. Means, S.D.s (n=10), and statistical significance are denoted; **P<0.01; ***P<0.001. Vector, transfected with an empty vector; KD, knockdown; OE, overexpression

Figure 6

MiR-125b regulates the activity of NF-κB signaling pathway by targeting A20 in NPC cells. (a) Western blot analysis showing the levels of p-IKKα/β, p-IκBα, p-p65(RelA), IKKα/β, IκBα, and p65 in the miR-125b mimic-transfected CNE2 cells, miR-125 inhibitor-transfected CNE2-IR cells, A20 KD CNE2 cells, A20 OE CNE2-IR cells, and their respective control cells. (b) Representative immunofluorescent staining showing the nuclear translocation of p-p65(RelA) in the miR-125b mimic-transfected CNE2 cells, miR-125 inhibitor-transfected CNE2-IR cells, A20 KD CNE2 cells, A20 OE CNE2-IR cells, and their respective control cells. (c) Western blot analysis showing p65(RelA) expression in the nuclear and cytoplasmic fractions of miR-125b mimic-transfected CNE2 cells, miR-125 inhibitor-transfected CNE2-IR cells, A20 KD CNE2 cells, A20 OE CNE2-IR cells, and their respective control cells. Nuc, nuclear fraction; Cyt, cytoplasmic fraction. (d) A luciferase reporter assay showing p65 transcriptional activity in the miR-125b mimic-transfected CNE2 cells, miR-125 inhibitor-transfected CNE2-IR cells, A20 KD CNE2 cells, A20 OE CNE2-IR cells, and their respective control cells. Three experiments were done; Means, S.D.s and statistical significance are denoted; ***P<0.001; ns, no significance. Vector, transfected with an empty vector; KD, knockdown; OE, overexpression

Figure 7

The effect of A20 expression changes on miR-125b-regulating NF-κB activity. Western blot analysis showing p-p65(RelA) levels (a), representative immunofluorescent staining showing the nuclear translocation of p-p65 (b), and luciferase reporter assay showing p65 transcriptional activity (c) in the A20 KD CNE2 cells transfected with miR-125b inhibitor, A20 OE CNE2-IR cells transfected with miR-125b mimic, and their respective control cells. Three experiments were done; Means, S.D.s and statistical significance are denoted; ***P<0.001. Vector, transfected with an empty vector; KD, knockdown; OE, overexpression

Figure 8

NF-κB mediates miR-125b/A20-regulating NPC cell proliferation and apoptosis. (a) Representative results (left) and statistical analyses (right) of EdU incorporation assay, plate clone formation assay and detection of cell apoptosis by flow cytometry in the A20 KD CNE2 cells transfected with plasmid expressing IκBα and control cells. (b) Representative results (left) and statistical analyses (right) of EdU incorporation assay, plate clone formation assay and detection of cell apoptosis by flow cytometry in the A20 KD CNE2 cells treated with BAY11-7082 and control cells. (c) Representative results (left) and statistical analyses (right) of EdU incorporation assay, plate clone formation assay and detection of cell apoptosis by flow cytometry in the A20 OE CNE2-IR cells transfected with plasmid expressing p65 and control cells. Three experiments were done; Means, S.D.s, and statistical significance are denoted; *P<0.05; ***P<0.001; ns, no significance. Vector, transfected with an empty vector; KD, knockdown; OE, overexpression