NF-κB inhibition reverses acidic bile-induced miR-21, miR-155, miR-192, miR-34a, miR-375 and miR-451a deregulations in human hypopharyngeal cells

Abstract

We previously demonstrated that acidic bile activates NF-κB, deregulating the expression of oncogenic miRNA markers, in pre-malignant murine laryngopharyngeal mucosa. Here, we hypothesize that the in vitro exposure of human hypopharyngeal cells to acidic bile deregulates cancer-related miRNA markers that can be reversed by BAY 11-7082, a pharmacologic NF-κB inhibitor. We repetitively exposed normal human hypopharyngeal primary cells and human hypopharyngeal keratinocytes to bile fluid (400 μmol/L), at pH 4.0 and 7.0, with/without BAY 11-7082 (20 μmol/L). We centred our study on the transcriptional activation of oncogenic miR-21, miR-155, miR-192, miR-34a, miR-375, miR-451a and NF-κB-related genes, previously linked to acidic bile-induced pre-neoplastic events. Our novel findings in vitro are consistent with our hypothesis demonstrating that BAY 11-7082 significantly reverses the acidic bile-induced oncogenic miRNA phenotype, in normal hypopharyngeal cells. BAY 11-7082 strongly inhibits the acidic bile-induced up-regulation of miR-192 and down-regulation of miR-451a and significantly decreases the miR-21/375 ratios, previously related to poor prognosis in hypopharyngeal cancer. This is the first in vitro report that NF-κB inhibition reverses acidic bile-induced miR-21, miR-155, miR-192, miR-34a, miR-375 and miR-451a deregulations in normal human hypopharyngeal cells, suggesting that acidic bile-induced events are directly or indirectly dependent on NF-κB signalling.

Keywords: BAY 11-7082; NF-κB; bile acids; hypopharyngeal cancer; miR-155; miR-192; miR-21; miR-34a; miR-375; miR-451a.

Figure 1

Luciferase assay demonstrates that 20 μmol/L of BAY 11‐7082 prevents the NF‐κB transcriptional activity in acidic bile‐treated normal human hypopharyngeal cells (HHPC) and human hypopharyngeal keratinocytes (HHK). A, Columns of graphs represent luciferase activity (mean ± standard error of three independent experiments) in (a) HHPC and (b) HHK, transfected with control luciferase reporter (luc2P) and NF‐κB luciferase responsive element (luc2P‐NF‐κB‐RE). B, Columns of graphs represent NF‐κB relative transcriptional activity (luc2P‐NF‐κB‐RE: NF‐κB luciferase responsive element/luc2P: control luciferase reporter) with/without BAY 11‐7082, in (a) HHPC and (b) HHK. Luc, luciferase

Figure 2

NF‐κB inhibitor (20 μmol/L ΒΑΥ 11‐7082) reverses the acidic bile‐induced deregulation of cancer‐related miRNA markers, in normal human hypopharyngeal primary cells (HHPC). A, Acidic bile induces in HHPC (a) an up‐regulation of the analysed “oncomirs” demonstrated by significantly higher miRNA levels, compared to controls, inverted by NF‐κB inhibitor (BAY 11‐7082). (b) Acidic bile induces a down‐regulation of the analysed “tumour suppressor” miRNAs, demonstrated by significantly lower expression levels, compared to controls, that is also inverted by NF‐κB inhibitor (BAY 11‐7082) in HHPC (one‐way ANOVA; by Friedman; *P < .05; **<P < .005; GraphPad Prism 6.0) B, Graphs depict the significantly (a) decreased expression levels of miR‐21, miR‐155 and miR‐192 and (b) increased expression levels of miR‐34a, miR‐375 and miR‐451a, in HHPC exposed to acidic bile with NF‐κB inhibitor (BAY 11‐7082), compared to HHPC exposed to acidic bile without NF‐κB inhibitor (P values by t test; mean ± SD; multiple comparisons by Holm‐Sidak; GraphPad Prism 6.0). (Normalization control: small RNA RNU6B). (Data were obtained from three independent experiments)

Figure 3

NF‐κB inhibitor (20 μmol/L ΒΑΥ 11‐7082) reverses the acidic bile‐induced deregulation of cancer‐related miRNA markers, in human hypopharyngeal keratinocytes (HHK). A, Acidic bile induces in HHK (a) an up‐regulation of the analysed “oncomirs”, demonstrated by significantly higher miRNA levels, compared to controls, inverted by NF‐κB inhibitor (BAY 11‐7082). (b) Acidic bile induces a down‐regulation of the analysed “tumour suppressor” miRNAs, demonstrated by significantly lower expression levels, compared to controls, that is also inverted by NF‐κB inhibitor (BAY 11‐7082) in HHK (one‐way ANOVA; by Friedman; *P < .05; **<P < .005; GraphPad Prism 6.0). B, Graphs depict the significantly (a) decreased expression levels of miR‐21, miR‐155 and miR‐192 and (b) increased expression levels of miR‐34a, miR‐375 and miR‐451a, in HHK exposed to acidic bile with NF‐κB inhibitor (BAY 11‐7082), compared to HHK exposed to acidic bile without NF‐κB inhibitor (P values by t test; mean ± SD; multiple comparisons by Holm‐Sidak; GraphPad Prism 6.0). (Normalization control: small RNA RNU6B). (Data were obtained from three independent experiments)

Figure 4

BAY 11‐7082‐induced miRNA phenotypes in acidic bile‐treated human hypopharyngeal primary cells (HHPC). A, NF‐κB inhibitor (BAY 11‐7082) in acidic bile‐treated HHPC induces a reversed phenotype of the analysed (a) “oncomirs” and (b) “tumour suppressor”, compared to neutral control and neutral bile (one‐way ANOVA; by Kruskal‐Wallis; *P < .05; GaphPad Prism 6.0). B, BAY 11‐7082 induces a reversed miRNA phenotype (with/without BAY 11‐7082) of each miR‐21, miR‐155, miR‐192, miR‐34a, miR‐375 and miR‐451a, in acidic bile exposure compared to other experimental‐ or control‐treated HHPC (one‐way ANOVA, by Kruskal‐Wallis; *P < .05; **P < .005; GraphPad Prism 6.0). C, (a) Acidic bile induces significantly higher miR‐21/375 ratios in HHPC, compared to neutral control or neutral bile. (b) BAY 11‐7082 induces significantly lower miR‐21/375 ratios (with/without BAY 11‐7082) in acidic bile‐treated HHPC, compared to neutral control or neutral bile (one‐way ANOVA, by Kruskal‐Wallis; *P < .05; **P < .005; GraphPad Prism 6.0). (Data were obtained from three independent experiments)

Figure 5

BAY 11‐7082‐induced miRNA phenotypes in acidic bile‐treated human hypopharyngeal keratinocytes (HHK). A, NF‐κB inhibitor (BAY 11‐7082) in acidic bile‐treated HHK induces a reversed phenotype (with/without BAY 11‐7082) of the analysed (a) “oncomirs” and (b) “tumour suppressor” miRNAs, compared to neutral controls or neutral bile. (one‐way ANOVA, by Kruskal‐Wallis; *P < .05; **P < .005; GraphPad Prism 6.0). B, BAY 11‐7082 induces a reversed miRNA phenotype (with/without BAY 11‐7082) of each miR‐21, miR‐155, miR‐192, miR‐34a, miR‐375 and miR‐451a, in acidic bile exposure compared to other experimental or control‐treated HHK. (one‐way ANOVA, by Kruskal‐Wallis; *P < .05; **P < .005; GraphPad Prism 6.0). C, (a) Acidic bile induces significantly higher miR‐21/375 ratios in HHK, compared to controls. (b) BAY 11‐7082 induces significantly lower miR‐21/375 ratios (with/without BAY 11‐7082) in acidic bile‐treated HHK, compared to controls (one‐way ANOVA, by Kruskal‐Wallis; *P < .05; **P < .005; GraphPad Prism 6.0). (Data were obtained from three independent experiments)

Figure 6

BAY 11‐7082 inhibits bile‐induced nuclear translocation of phospho‐NF‐κB and phospho‐STAT3 in normal human hypopharyngeal cells. Immunofluorescence staining of (A) phospho‐NF‐κB (p‐p65 S536) and (B), phospho‐STAT3 (Tyr705) reveals that application of BAY 11‐7082 particularly inhibits the p‐NF‐κB and p‐STAT3 nuclear translocation in acidic bile‐treated human hypopharyngeal primary cells (HHPC), demonstrating decreased p‐p65 and p‐STAT3 nuclear staining [green: p‐p65 (S536) or pSTAT3 (Tyr705); blue: DAPI for nuclear staining]

Figure 7

Acidic bile‐induced interactions between NF‐κB and oncogenic miR‐21, ‐155, ‐192, ‐34α, ‐375 and ‐451α markers, in treated human hypopharyngeal primary cells and their role in hypopharyngeal carcinogenesis