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.

Biliary reflux as a causal factor in hypopharyngeal carcinoma: New clinical evidence and implications

“…Because PI3K activation may lead to downstream NF-κB activation, 35 its role in bile-induced NF-κB activation requires further exploration. 9,33,34,43,44 Bile(+) tumors also demonstrate an intense downregulation of tumor suppressors miR-489, miR-504, and miR-99a compared with their ANTs, a finding that is in keeping with previous studies suggesting their involvement in the initiation and progression of HSCC. 46,47 The data from the current study do not fully support a strong association between the NOTCH family and bile(+) HSCC.…”

“…It is apparent that activation of NF-κB is a common finding in HSCC from patients with documented biliary-esophageal reflux. [4][5][6][7][8][9]16,[32][33][34][43][44][45] In aggressive bile(+) HSCC, there appears to be a selective upregulation of NF-κB transcriptional factors RELA(p65) and c-REL; antiapoptotic bcl-2; and oncogenic EGFR, STAT3, and WNT5A, which is consistent 14,15,29 Clinical observations described herein further suggest that in bile(+) HSCC, constitutive activation of NF-κB also is capable of producing a cancer-related mRNA and miRNA phenotype similarly found in our preclinical in vitro and in vivo models and negatively affected by NF-κB inhibition ( Table 2).…”

“…34,43 Bile(+) HSCC produces higher transcriptional levels of PIK3CA, TP53, NOTCH1, NOTCH2, and NOTCH3 compared with their ANTs or bile(-) tumors. 34,43 Bile(+) HSCC produces higher transcriptional levels of PIK3CA, TP53, NOTCH1, NOTCH2, and NOTCH3 compared with their ANTs or bile(-) tumors.…”

“…We performed miRNA analysis using qPCR with specific primers for target miRNAs of the human genome and normalization control small nuclear RNA RNU6B (snRNA RNU6-2) (miScript Primer Assay and miScript SYBR Green PCR Kit; Qiagen) as described previously 34 and demonstrated in Supporting Table S1B. Relative miRNA expression of tumor to miRNAs of ANTs was estimated using CFX96 software (Bio-Rad) for each specific miRNA marker.…”

MiR‐802 causes nephropathy by suppressing NF‐κB‐repressing factor in obese mice and human

“…27,28 These inhibitors could improve NF-κB-mediated inflammation and tissue damages in mouse and human studies. 27,28 In 1999, Nourbakhsh et al described the functional characterization of NRF (NF-κB-repressing factor), which abolishes the transcriptional activity of the bordering NF-κB binding sites. 24 Then, lots of studies continued to explore the biological roles of NRF in different molecular signalling.…”