<i>Helicobacter pylori</i> Initiates a Mesenchymal Transition through ZEB1 in Gastric Epithelial Cells

<div><p>Chronic <i>Helicobacter pylori</i> infection provokes an inflammation of the gastric mucosa, at high risk for ulcer and cancer development. The most virulent strains harbor the <i>cag</i> pathogenicity island (<i>cag</i>PAI) encoding a type 4 secretion system, which allows delivery of bacterial effectors into gastric epithelial cells, inducing pro-inflammatory responses and phenotypic alterations reminiscent of an epithelial-to-mesenchymal transition (EMT). This study characterizes EMT features in <i>H. pylori</i>-infected gastric epithelial cells, and investigates their relationship with NF-κB activation. Cultured human gastric epithelial cell lines were challenged with a <i>cag</i>PAI<i>+ H. pylori</i> strain or <i>cag</i> isogenic mutants. Morphological changes, epithelial and mesenchymal gene expression and EMT-related microRNAs were studied. <i>H. pylori</i> up-regulates mesenchymal markers, including ZEB1. This transcription factor is prominently involved in the mesenchymal transition of infected cells and its up-regulation depends on <i>cag</i>PAI and NF-κB activation. ZEB1 expression and NF-κB activation were confirmed by immunohistochemistry in gastric mucosa from <i>cag</i>PAI<i>+ H. pylori</i>-infected patients. Gastric epithelial cell lines express high miR-200 levels, which are linked to ZEB1 in a reciprocal negative feedback loop and maintain their epithelial phenotype in non-infected conditions. However, miR-200b/c were increased upon infection, despite ZEB1 up-regulation and mesenchymal morphology. In the miR-200b-200a-429 cluster promoter, we identified a functional NF-κB binding site, recruiting NF-κB upon infection and trans-activating the microRNA cluster transcription. In conclusion, in gastric epithelial cells, <i>cag</i>PAI+ <i>H. pylori</i> activates NF-κB, which transactivates ZEB1, subsequently promoting mesenchymal transition. The unexpected N-FκB-dependent increase of miR-200 levels likely thwarts the irreversible loss of epithelial identity in that critical situation.</p> </div

MiR-200 regulate ZEB1 expression in basal conditions and are up-regulated by <i>H. pylori</i>.

<p>(A) Cell morphology observed by phase contrast microscopy of AGS cells treated for 5 day with 100 nM antisense (as200b/c) or scrambled (sc200) oligonucleotides (scale bar, 20 µm). (B) ZEB1 immunofluorescence in the same conditions (scale bar, 20 µm) (C) RT-qPCR data of ZEB1 mRNA in the same conditions. Bars represent the mean ± SD of ZEB1 mRNA relative to HPRT mRNA compared to non transfected cells (n = 5; * p<0.05). (D) ZEB1 and tubulin immunoblots in the same conditions. (E) RT-qPCR data of mature miR-200b and -200c in AGS cells infected or not (NI) with either wt <i>H. pylori</i> (WT) or isogenic mutants (ΔCagA, ΔCagE) at MOI 100 bacteria/cells for 24 h. Bars indicate mean ± SD of miRNA expression normalized to U6 snRNA and compared to NI (n = 6; ** p<0.01). (F) RT-qPCR data of primary miR-200b-200a-429 transcript in the same infection conditions. Bars indicate mean ± SD of pri-miRNA expression normalized to HPRT1 and compared to NI (n = 4; * p<0.05, *** p<0.01).</p

<i>H. pylori</i>, ZEB1, p65 NF-κB, E-cadherin immunostaining and miR-200b <i>in situ</i> hybridization in non infected human gastric mucosa (left pannels) or mucosa infected with <i>cag</i>PAI+ <i>H. pylori</i> (right pannels).

<p>Images are representatives of the detection by immunohistochemistry coupled to peroxydase activity (in brown) of <i>H. pylori</i> in the lumen of gastric glands at the apical surface of gastric epithelial cells, which display an intense ZEB1 and p65 expression mainly in the nucleus, despite a similar E-cadherin expression and localization at cell/cell junction in the <i>H. pylori</i> infected specimen and the non-infected one. MiR-200b detected by ISH coupled to phosphatase alkaline activity (in dark blue) is highly expressed in gastric glands of <i>H. pylori</i>–infected case. Typical images of the same case out of three infected patients and the same case out of three uninfected cases are shown. Bar, 50 µm.</p

Changes in expression of mesenchymal and epithelial markers in AGS, MKN74 and NCI-N87 cells infected for 24 h with the H. pylori strain 26695.

<p>Data represent RT-qPCR results of the individual genes normalized to that of HPRT and compared to non infected cells (mean, n = 3, *p<0.05, **p<0.01, ***p<0.001).</p

NF-κB-dependent mesenchymal phenotype of infected AGS cells.

<p>In all experiments, AGS cells were infected or not (NI) with <i>cagPAI+ H. pylori</i> (WT) at MOI 100 bacteria/cells for 24 h. (A) Activities of SV40 promoter (pGL3-p), or <i>miR-200b-200a-429</i> promoter wild type (pGL3-prom200b) or mutated on the NF-κB site (pGL3-prom200b mut); bars represent mean ± SD of relative luciferase activities of each promoter reporter normalized to that of NI pGL3-p transfected cells (n = 3; ** p<0.01). (B) NF-κB activation upon infection (WT) in cells transfected either with pEGFP or with pEGFP-IκB. Bars represent mean ± SD of relative NF-κB reporter luciferase activity compared to NI pEGFP-transfected cells (n = 3; *p<0.05). (C) Cell morphology observed by phase contrast microscopy, in the same conditions. Bar, 20 µm. (D) RT-qPCR data of ZEB1 and pri-miR-200b-200a-429 in pEGFP- or pEGFP-IκB-transfected cells. Bars indicate mean ± SD of RNA expression normalized to HPRT1 and compared to NI (n = 3, * p<0.05, *** p<0.001). (E) <i>MiR-200b-200a-429</i> promoter activities measured as in (A) in cells transfected either with pEGFP or with pEGFP-IκB. (F) Chromatin immunoprecipitation assays using anti-NF-κB antibody on the promoters of miR-200b (prom200b), IL-8 (promIL-8) or ZEB1 (promZEB1). Bars represent NF-κB enrichment on a given promoter in either uninfected or infected cells, calculated as the following ratio: 2^{−ΔCt IPNF-κB}/2^{−ΔCt controlIP}, with ΔCt = Ct IP (with NF-κB antibody or without (control)) – Ct input (input corresponds to chromatin before immunoprecipitation).</p

Kinetics of changes in IL-8 induction (A), ZEB1 and pri-miR-200b (B) and E-cadherin and vimentin (C) expressions upon infection.

<p>Cells were infected with either wt <i>H. pylori</i> (WT) or its <i>cagA</i>-deletion isogenic mutant (ΔCagA) at MOI 100 bacteria/cells for the indicated period of times. Bars indicate the fold changes of the individual genes upon infection (mean of duplicates ± SD of RNA expression normalized to HPRT1 and compared to NI).</p

Changes in mesenchymal and epithelial gene expression and miR-200 levels. 10 days post-infection with cagPAI+ H. pylori (Hp WT) or the isogenic CagA-deficient strain.

<p>48 hrs post-infection at a MOI 100, infected and non-infected cells were trypsinized and subcultured for 10 days in a 6-well plate starting at an initial cell density of 2,000 cells/well. The culture medium was changed every other day. Data represent mean ± SD of RTqPCR results of the individual genes or miRNAs relative to HPRT1 or snoR25, respectively, and compared to non infected cells; n = 4; *: p-value <0.05, **: p-value <0.01, ***: p-value <0.001.</p

<i>H. pylori</i> up-regulates ZEB1 in gastric epithelial cells.

<p>(A) RT-qPCR data of ZEB1 mRNA upon 24 h infection with wild type <i>H. pylori</i> (Hp WT) or its isogenic mutants deleted either for <i>cagA</i> (Hp Δ<i>cag</i>A) or <i>cagE</i> (Hp Δ<i>cag</i>E); bars represent the mean ± SD of ZEB1 mRNA relative to HPRT mRNA compared to non infected cells (NI) (n = 5; * p<0.05). (B) ZEB1 immunofluorescence in non-infected cells (NI) or upon infection as in (A); bar, 40 µm. (C) Cell morphology observed by phase contrast microscopy of AGS cells transfected with siZEB1 or control siRNA (20 nM) prior infection as in (A). Bar, 40 µm. (D) ZEB1 or α-tubulin immunoblots in cells treated with siZEB1 or control siRNA and infected as in (A).</p

Targeting the Production of Oncogenic MicroRNAs with Multimodal Synthetic Small Molecules

MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and revealed to be oncogenic and to play a pivotal role in initiation and progression of these pathologies. It is now clear that the inhibition of oncogenic miRNAs, defined as blocking their biosynthesis or their function, could find an application in the therapy of different types of cancer in which these miRNAs are implicated. Here we report the design, synthesis, and biological evaluation of new small-molecule RNA ligands targeting the production of oncogenic microRNAs. In this work we focused our attention on miR-372 and miR-373 that are implicated in the tumorigenesis of different types of cancer such as gastric cancer. These two oncogenic miRNAs are overexpressed in gastric cancer cells starting from their precursors pre-miR-372 and pre-miR-373, two stem-loop structured RNAs that lead to mature miRNAs after cleavage by the enzyme Dicer. The small molecules described herein consist of the conjugation of two RNA binding motives, i.e., the aminoglycoside neomycin and different natural and artificial nucleobases, in order to obtain RNA ligands with increased affinity and selectivity compared to that of parent compounds. After the synthesis of this new series of RNA ligands, we demonstrated that they are able to inhibit the production of the oncogenic miRNA-372 and -373 by binding their pre-miRNAs and inhibiting the processing by Dicer. Moreover, we proved that some of these compounds bear anti-proliferative activity toward gastric cancer cells and that this activity is likely linked to a decrease in the production of targeted miRNAs. To date, only few examples of small molecules targeting oncogenic miRNAs have been reported, and such inhibitors could be extremely useful for the development of new anticancer therapeutic strategies as well as useful biochemical tools for the study of miRNAs’ pathways and mechanisms. Furthermore, this is the first time that a design based on current knowledge about RNA targeting is proposed in order to target miRNAs’ production with small molecules

Encapsidation of RNA–Polyelectrolyte Complexes with Amphiphilic Block Copolymers: Toward a New Self-Assembly Route

Amphiphilic block copolymers are molecules composed of hydrophilic and hydrophobic segments having the capacity to spontaneously self-assemble into a variety of supramolecular structures like micelles and vesicles. Here, we propose an original way to self-assemble amphiphilic block copolymers into a supported bilayer membrane for defined coating of nanoparticles. The heart of the method rests on a change of the amphiphilicity of the copolymer that can be turned off and on by varying the polarity of the solvent. In this condition, the assembly process can take advantage of specific molecular interactions in both organic solvent and water. While the concept potentially could be applied to any type of charged substrates, we focus our interest on the design of a new type of polymer assembly mimicking the virus morphology. A capsid-like shell of glycoprotein-mimic amphiphilic block copolymer was self-assembled around a positively charged complex of siRNA and polyethyleneimine. The process requires two steps. Block copolymers first interact with the complexes dispersed in DMSO through electrostatic interactions. Next, the increase of the water content in the medium triggers the hydrophobic effect and the concomitant self-assembly of free block copolymer molecules into a bilayer membrane at the complex surface. The higher gene silencing activity of the copolymer-modified complexes over the complexes alone shows the potential of this new type of nanoconstructs for biological applications, especially for the delivery of therapeutic biomolecules