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.
Highlights
Hypopharyngeal cancer is one of the most aggressive subtypes of head and neck squamous cell carcinoma (HNSCC).[1]
Human hypopharyngeal primary cells exposed to acidic bile with BAY 11-7082 demonstrated a significant decrease in “oncomirs” miR21 (P = .006), miR-155 (P = .0035) and of miR-192 levels (P < .00001) (Figure 2B-a), as well as a significant increase in “tumour suppressor” miR-34a (P = .00023), miR-375 (P = .00134) and of miR-451a levels (P < .000001) (Figure 2B-b), compared to HHPC exposed to acidic bile without NF-jB inhibitor (t test analysis; multiple comparisons by Holm-Sidak)
We further showed HHPC treated with acidic bile without BAY 11-7082 demonstrated a significantly higher miR-21/375 ratio, compared to neutral control or neutral bile, (P = .0022, and P = .0415, respectively, by Kruskal-Wallis) (Figure 4B), and hypopharyngeal keratinocytes (HHK) treated with acidic bile without NF-jB inhibitor showed a significantly higher miR-21/375 ratio compared to neutral control or acid alone (P = .0022 and P = .0415, respectively, by Kruskal-Wallis)
Summary
Hypopharyngeal cancer is one of the most aggressive subtypes of head and neck squamous cell carcinoma (HNSCC).[1]. | 2923 between acidic bile or GDF and early pre-neoplastic events in laryngopharyngeal mucosa.[8,9,10] the combination of bile and acid (pH ≤4.0) constitutively activates NF-jB, up-regulating the expression of cancer-related genes and deregulating the expression of oncogenic miRNA markers, such as “oncomirs” miR-21, miR-192, miR-155 and “tumour suppressors” miR-34a, miR-375 and miR-451a, in pre-malignant lesions of murine laryngopharyngeal mucosa.[9,10] Here, we describe an in vitro model exploring repetitive exposures of normal human hypopharyngeal cells to acidic bile, with and without BAY 11-7082, a pharmacologic inhibitor of NF-jB.[11] We hypothesize that NF-jB inhibitor is capable of preventing the acidic bile-induced up-regulation of “oncomirs” miR-21, miR-155 and miR192 and down-regulation of “tumour suppressor” miR-34a, miR-375 and miR-451a, previously associated with laryngopharyngeal cancer,[12,13,14,15,16,17] providing insight into interactions of transcriptionally active NF-jB with cancer-related miRNA markers. Evidence that inhibition of acidic bile-induced NF-jB activation effectively reverses the altered cancer-related miRNA phenotype will encourage the in vivo application of NF-jB inhibitors, as possible preventers of acidic bile effect in hypopharyngeal mucosa
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