Anti‑inflammatory role of microRNA‑429 in human gingival epithelial cells‑inhibition of IL‑8 production through direct binding to IKKβ mRNA

Abstract

MicroRNAs (miRNAs), a family of small non‑coding RNAs, serve a pivotal role in the regulation of the inflammation by modulating the expression of various genes. However, the molecular mechanism by which miRNAs regulate inflammation‑associated molecules in oral epithelial cells remains to be elucidated. The present study examined the biological function of miR‑429 by performing the gain‑/loss‑of‑function studies of miR‑429 in a gingival squamous cell carcinoma line Ca9‑22 cells that either over‑ or under‑expressed miR‑429 through transient transfection with miR‑429 mimic or miR‑429 inhibitor, respectively. The results demonstrated that the over‑expression of miR‑429 suppressed the mRNA level of several interleukins, including IL‑8. In addition, the over‑expression of miR‑429 reduced IL‑8 secretion under the basal and TNF‑α stimulated conditions, whereas the secretion of IL‑8 was enhanced when miR‑429 was under‑expressed. The over‑expression of miR‑429 inhibited the activation of the transcription factor NF‑κB. Furthermore, we found that miR‑429 suppressed both mRNA and protein levels of IKKβ via its direct binding to the 3'‑untranslated region of IKKβ mRNA. In addition, the downregulation of IKKβ by small interfering RNA reduced both NF‑kB activity and IL‑8 production in Ca9‑22 cells. Taken together, the findings revealed the molecular mechanism of miR‑429 to regulate the inflammatory mediator in gingival cells and suggested that it could be useful as a therapeutic target of oral inflammatory diseases.

Keywords: IKKβ; IL‑8; NF‑κB; gingival epithelial cell; inflammation; microRNA‑429.

Figure 1.

Over- and under-expression of miR-429 in Ca9-22 cells. Ca9-22 cells were transfected with (A) miR-429 mimic or NCm and with (B) miR-429 inhibitor or NCi or (C) stimulated with TNF-α (5, 10 or 50 ng/ml). The expression level of miR-429 was determined by reverse transcription-quantitative PCR. U6 was used as a housekeeping gene control. Data are presented as the means ± standard deviation (n=3). The t-test or one-way ANOVA followed by Bonferroni's multiple comparison test was used for the calculation of P-values. **P<0.01, ***P<0.001 vs. the control group. miR, microRNA; mimic, miR-429 mimic; NCm, negative control mimic; inhibitor, miR-429 inhibitor; NCi, negative control inhibitor.

Figure 2.

The effect of over- and under-expression of miR-429 on the expression of proinflammatory cytokines in Ca9-22 cells. Expression levels of proinflammatory cytokines, (A) IL-1β, (B) IL-6 and (C) IL-8, in Ca9-22 cells transfected with miR-429 mimic or NCm were determined by reverse transcription-quantitative PCR. Expression levels of proinflammatory cytokines, (D) IL-1β, (E) IL-6 and (F) IL-8, in Ca9-22 cells transfected with miR-429 inhibitor or NCi were also determined. GAPDH was used as a housekeeping gene control. Data are presented as the means ± standard deviation (n=3). The t-test was used for the calculation of P-values. *P<0.05, **P<0.01 vs. the control group. miR, microRNA; mimic, miR-429 mimic; NCm, negative control mimic; inhibitor, miR-429 inhibitor; NCi, negative control inhibitor.

Figure 3.

The effect of over- and under-expression of miR-429 on the secretion of IL-8 in Ca9-22 cells. Following the transfection of miR-429 mimic, NCm, miR-429 inhibitor or NCi, the cells were cultured with or without TNF-α (50 ng/ml). The concentration of IL-8 secreted in the medium was measured by ELISA. The amount of IL-8 derived from the cells transfected with the (A) miR-429 mimic or (B) miR-429 inhibitor was compared with that of the corresponding NC group. The IL-8 production was compared between the conditions with or without TNF-α stimulation under the same experimental conditions. Data are presented as the means ± standard deviation (n=3). Two-way ANOVA followed by Bonferroni's multiple comparison test was used for the calculation of P-values. ^&&P<0.01 vs. the control group. ^##P<0.01 vs. no TNF-α stimulation group. miR, microRNA; mimic, miR-429 mimic; NCm, negative control mimic; inhibitor, miR-429 inhibitor; NCi, negative control inhibitor.

Figure 4.

The effect of over- and under-expression of miR-429 on the activation of NF-kB in Ca9-22 cells. The expression level of NF-kB p65 protein was measured by Western blot analysis after the incubation of Ca9-22 cells, transfected with (A) miR-429 mimic or NCm and with (B) miR-429 inhibitor or NCi, with or without the subsequent stimulation of TNF-α (50 ng/ml). The levels of p-p65 and p65 were normalized by GAPDH level and the relative p-p65/p65 level was calculated. Data are presented as the means ± standard deviation (n=3). The luciferase activity in the cells was determined by dual luciferase reporter assay system after the co-transfection of the plasmids with (C) miR-429 mimic or NCm and with (D) miR-429 inhibitor or NCi. Data are presented as the means ± standard deviation (n=4). Two-way ANOVA followed by Bonferroni's multiple comparison test was used for the calculation of P-values. ^&P<0.05, ^&&P<0.01 vs. the control group. ^##P<0.01 vs. no TNF-α stimulation group. miR, microRNA; mimic, miR-429 mimic; NCm, negative control mimic; inhibitor, miR-429 inhibitor; NCi, negative control inhibitor; p-, phosphorylated.

Figure 5.

miR-429 direct binding to IKKβ in Ca9-22 cells. (A) The putative binding site of miR-429 on IKKβ-WT and its corresponding site on IKKβ-MUT. The cells were transfected with miR-429 mimic, miR-429 inhibitor or the corresponding NC. The levels of IKKβ (B and C) mRNA and (D) protein following the treatment with miR-429 mimic and with miR-429 inhibitor are shown. The IKKβ mRNA and protein levels were determined by reverse transcription-quantitative PCR and western blot analysis, respectively. GAPDH was used as a housekeeping gene control. (E and F) The luciferase assay in Ca9-22 cells co-transfected with IKKβ (IKKβ-WT or IKKβ-MUT) and (E) miR-429 mimic or NCm and (F) miR-429 inhibitor or NCi. The luciferase activity was determined by dual luciferase reporter assay system. Data are presented as the means ± standard deviation (n=3). The t-test was used for the calculation of P-values. *P<0.05, **P<0.01 vs. the control group. miR, microRNA; WT, wild type; MUT, mutant type; mimic, miR-429 mimic; NCm, negative control mimic; inhibitor, miR-429 inhibitor; NCi, negative control inhibitor.

Figure 6.

Effect of siRNA-IKKβ on IL-8 and NF-kB levels in Ca9-22 cells. The expression of (A) IKKβ and (B) IL-8 mRNA was analyzed by reverse transcription-quantitative PCR and GAPDH was used as a housekeeping gene control. (C) The expression of IKKβ protein. (D) The levels of p-p65 and p65 are shown after the treatment with siRNA-IKKβ or NCsi, with or without TNF-α stimulation (50 ng/ml). The protein level was measured by western blot analysis. The levels of IKKβ, p-p65 and p65 were normalized by GAPDH level and the relative p-p65/p65 level was calculated. (E) IL-8 secretion from Ca9-22 cells transfected with siRNA-IKKβ or NCsi, with or without TNF-α stimulation (50 ng/ml). The concentration of IL-8 secreted in the medium was measured by ELISA. Data are presented as the means ± standard deviation (n=3). **P<0.01, ***P<0.001 (Student's t-test), ^&P<0.05, ^&&P<0.01 (two-way ANOVA followed by Bonferroni's multiple comparison test) vs. the control group. ^##P<0.01 (two-way ANOVA followed by Bonferroni's multiple comparison test) vs. the no TNF-α stimulation group. siRNA-IKKβ, small interfering RNA targeting IKKβ; p-, phosphorylated; NCsi, negative control siRNA.