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.

Figure 1

GDF-induced preneoplastic lesions in murine LM of C57BL/6J mice (H&E staining). (A) Normal LM: intermediate (low squamoid nonkeratinizing) epithelium. (B) Hyperplastic LM: thickened intermediate epithelium. (C) Abnormal hyperplastic/mildly dysplastic LM: thickened intermediate epithelium, hyperchromatic or pleomorphic basal cells expanding in the stratum spinosum. (D) Moderately dysplastic LM: high degree of basal layer expansion, nuclear hyperchromatism with increase of nuclear to cytoplasmic ratios, and loss of cell polarity. (E) Columns of graph created by GraphPad software correspond to mean thickness of LM under different treatments allowing comparison to control (*P < .05; **P < .005 ANOVA, Kruskal-Wallis test; GraphPad Prism 6.0). (F) Graph demonstrating the percentage of C57Bl/6J mice exhibiting histological alterations in LM.

Figure 2

GDF-induced NF-κB activation in murine LM. (A) Immunohistochemical analysis for p-NF-κB (p65 S536) (from left to right): control (normal) untreated LM, cytoplasmic staining; glucose-treated LM, weak cytoplasmic or nuclear staining sporadically of few basal cells; acid alone–treated LM, nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers; hyperplastic/dysplastic neutral bile–, acidic bile–, DCA-, and CDCA-treated LM, intense nuclear and cytoplasmic staining of cells of basal and parabasal/suprabasal layers. (B) Image analysis algorithm(s) (red, nuclear positive staining of p-NF-κB; orange, intense positive cytoplasmic staining of p-NF-κB; yellow, weak cytoplasmic staining of p-NF-κB; blue, negative p-NF-κB staining). [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems), which generated algorithm(s) illustrating the mucosal and cellular compartments demonstrating p-NF-κB staining].

Figure 3

GDF induces molecular alterations underlying preneoplastic alterations of murine LM related to NF-κB activation, cell proliferation (indicated by increased ΔNp63, Ki67, and CK14 levels), EMT (supported by changes of cell-cell adhesion molecules E-cadherin and β-catenin), and STAT3 activation. IF staining (A) and AQUA (B) for (a) p-NF-κB (p65 S536) (green) and ΔNp63 (red); (b) Ki67 (green) and E-cadherin (red); (c) CK14 (green) and ϐ-catenin (red); and (d) p-STAT3 (Tyr705) (green) (from left to right): Normal untreated LM, glucose- and acid alone–treated LM; and hyperplastic/dysplastic neutral bile–, acidic bile–, DCA-, and CDCA-treated LM derived from C57Bl/6J mice. DAPI (blue) was used for nuclear staining (DyLight®488 for green and DyLight®549 for red). The box plots represent the mean ranks. The upper line indicates the highest value, the lower line the lowest value, and the middle line the mean of AQUA normalized quantities of each variable. Statistically significant difference between AQUA score means is indicated for GDF-treated LM versus normal untreated LM (right) and GDF- versus acid-treated LM (left) (*P < .05; **P < .005; ***P < .0005; one-way ANOVA, Kruskal-Wallis and Dunn’s multiple comparison tests; GraphPad Prism 6.0).

Figure 4

Correlation among GDF-induced molecular alterations in treated LM by Pearson. (A) Diagrams depicting positive correlation between (a) NF-κB and ΔNp63 or p-STAT3, (b) NF-κB and β-catenin levels, (c) ΔNp63 and CK14 or p-STAT3 levels, and (d) p-STAT3 and β-catenin levels in treated LM (P value < .05). (B) Graph demonstrates strong inverted phenotype of cell adhesion molecules β-catenin and E-cadherin in acidic bile–treated LM versus untreated LM (P value < .0001).

Figure 5

In vivo acidic bile induced deregulation of cancer-related miRNAs in murine LM. (A) Upregulation of “oncomirs” miR-21, miR-155, and miR-192 in acidic bile–treated LM versus untreated LM (by one-way ANOVA). (B) Downregulation of tumor suppressors miR-375, -34a, and -451a in GDF and particularly in acidic bile–treated LM versus untreated LM (by one-way ANOVA). (C) Graphs represent the deregulation of the analyzed “oncomirs” and tumor suppressor miRNAs in each GDF group of treatment. We show a significantly higher upregulation of “oncomirs” and more intense downregulation of tumor suppressor miRNAs in acidic bile–treated LM compared with LM exposed to neutral bile/DCA/CDCA (ANOVA, by Kruskal-Wallis). Columns of graphs created by Graph Pad Prism software 6.0 correspond to miRNA means. The upper line of column indicates the highest measurement (*P < .05; **P < .005; ***P < .0005;****P < .00005).

Figure 6

Correlation among GDF-induced deregulation of cancer related miRNAs and NF-κB activation. Diagrams depicting (A) positive correlation between (a) miR-21, -155, and -192 and (b) between miR-155 and -192 levels (miRNA levels normalized to RNU6); (B) positive correlation between NF-κB activation and “oncomirs” miR-21, -155, and -192 expression; and (C) an inverse correlation between acidic bile–induced NF-κB activation and tumor suppressors (a) miR-34a, miR-375, and (b) miR-451a expression in treated LM (P value < .0001; by Pearson).