miR-21, miR-155, miR-192, and miR-375 Deregulations Related to NF-kappaB Activation in Gastroduodenal Fluid–Induced Early Preneoplastic Lesions of Laryngeal Mucosa In Vivo

Abstract

Gastroduodenal refluxate found in the upper aerodigestive tract is not clinically uncommon. We recently demonstrated the neoplastic potential of gastroduodenal fluids (GDF) on hypopharyngeal mucosa, via NF-κB, using in vitro and in vivo models. Here we will explore the in vivo effect of GDF on laryngeal mucosa (LM) to induce early preneoplastic lesions related to NF-κB activation, along with deregulation of specific microRNA (miRNA) markers previously linked to laryngeal cancer. We used histological, immunohistochemical, automated quantitative analysis and quantitative polymerase chain reaction to examine LM from 35 C57Bl/6J mice previously treated with topical GDF against corresponding controls (4 experimental and 3 control groups; 5 mice/group). Our analysis showed that GDF produced early preneoplastic lesions in treated LM related to NF-κB activation. LM treated by acid and bile combination demonstrated significantly higher expression of the analyzed cell proliferation markers (Ki67, CK14, ∆Np63), oncogenic p-STAT3, and changes of cell adhesion molecules (E-cadherin, ϐ-catenin) versus untreated LM or LM exposed to acid alone (P < .0005). Furthermore, acidic bile but not neutral bile appeared to accelerate the expression of “oncomirs” miR-21, miR-155, and miR-192 (acidic bile versus neutral bile, P < .0001), while reducing tumor suppressor miR-375 (acidic bile versus neutral bile, P = .0137), previously linked to NF-κB and laryngeal cancer. Finally, acidic bile induced reduction of miR-34a, miR-375, and miR-451a, exhibiting an inverse correlation with NF-κB activation. SIGNIFICANCE: Bile in combination with acid has a selective tumorigenic effect on LM, inducing deregulation of “oncomirs” and tumor suppressor miRNAs, produced by NF-κB activation with molecular and early histopathological alterations linked to neoplastic transformation. Systematic acid suppression may in part convey a protective role.

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

“…Others have shown that constitutive up‐regulation of miR‐155 may mediate prolonged inflammatory reactions leading to cancer 18, 52. We previously showed a significant up‐regulation of miR‐155 in laryngopharyngeal mucosa treated by acidic bile accompanied by pre‐neoplastic lesions 10. Our current findings show that BAY 11‐7082‐induced miR‐155 levels resulted in strong positive correlations with pro‐inflammatory TNF‐α, IL‐1β and ΙL‐6 mRNAs, suggesting a protective role of NF‐κB inhibition in pro‐inflammatory events linked to downstream oncogenic pathways.…”

“…Our current findings demonstrate that, among the analysed miRNA markers, miR‐21 and miR‐375 are the most affected by the NF‐κB inhibitor, underscoring the role of activated NF‐κB with miR‐21 and miR‐375, in promoting acidic bile‐induced cancer‐related molecular alterations in hypopharyngeal cells. There is further evidence that microRNA markers, such as “oncomir” miR‐21 and “tumour suppressor” miR‐375, play a crucial role in initiation and progression of HNSCC 10, 12, 13. Arantes LMRB et al recently reported the fundamental role of miR‐21, as a biomarker, in head and neck carcinogenesis,47 while miR‐375 has been proposed as a predictive biomarker for early diagnosis in laryngeal cancer 48…”

“…Recent in vitro and in vivo studies have defined NF‐κB to be a possible mechanistic link between acidic bile or GDF and early pre‐neoplastic events in laryngopharyngeal mucosa 8, 9, 10. Specifically, the combination of bile and acid (pH ≤4.0) constitutively activates NF‐κB, 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‐κB 11.…”

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

“…[21][22][23][24][25][26] An independent association has been demonstrated between NF-κB activation and upregulation of oncogenic miR-21 and/ or downregulation of the tumor suppressors miR-34a and miR-451a. 7,8 To explore our hypothesis, we performed a pilot study analyzing a group of HSCC specimens and their corresponding adjacent normal tissues (ANTs) derived from patients with documented bile reflux compared with controls to identify possible differences in expression levels of activated NF-κB and related mRNA and miRNA phenotypes. 29 We hypothesized that invasive squamous cell carcinoma derived from patients with documented bile reflux may demonstrate strong NF-κB activation accompanied by messenger RNA (mRNA) and miRNA phenotypes previously identified in murine hypopharyngeal mucosa chronically exposed to acidic bile.…”

The functional role of microRNAs in laryngeal carcinoma

“…The elevated expression of miR-155 is associated with downregulation of SOCS1 and increased STAT3 expression. The activation of STAT3 can promote the development and differentiation of laryngeal squamous cell carcinoma [33]. This suggests that miR-155 exerts a carcinogenic effect by regulating the expression of the target gene SOCSl-STAT3.…”