MIR106B and MIR93 Prevent Removal of Bacteria From Epithelial Cells by Disrupting ATG16L1-Mediated Autophagy

doi:10.1053/j.gastro.2013.09.006

Gastroenterology

Volume 146, Issue 1, January 2014, Pages 188-199

https://doi.org/10.1053/j.gastro.2013.09.006 Get rights and content

Background & Aims

Variants in genes that regulate autophagy have been associated with Crohn's disease (CD). Defects in autophagy-mediated removal of pathogenic microbes could contribute to the pathogenesis of CD. We investigated the role of the microRNAs (miRs) MIR106B and MIR93 in induction of autophagy and bacterial clearance in human cell lines and the correlation between MIR106B and autophagy-related gene 16L1 (ATG16L1) expression in tissues from patients with CD.

Methods

We studied the ability of MIR106B and MIR93 to regulate ATG transcripts in human cancer cell lines (HCT116, SW480, HeLa, and U2OS) using luciferase report assays and bioinformatics analyses; MIR106B and MIR93 mimics and antagonists were transfected into cells to modify levels of miRs. Cells were infected with LF82, a CD-associated adherent-invasive strain of Escherichia coli, and monitored by confocal microscopy and for colony-forming units. Colon tissues from 41 healthy subjects (controls), 22 patients with active CD, 16 patients with inactive CD, and 7 patients with chronic inflammation were assessed for levels of MIR106B and ATG16L1 by in situ hybridization and immunohistochemistry.

Results

Silencing Dicer1, an essential processor of miRs, increased levels of ATG protein and formation of autophagosomes in cells, indicating that miRs regulate autophagy. Luciferase reporter assays indicated that MIR106B and MIR93 targeted ATG16L1 messenger RNA. MIR106B and MIR93 reduced levels of ATG16L1 and autophagy; these increased after expression of ectopic ATG16L1. In contrast, MIR106B and MIR93 antagonists increased formation of autophagosomes. Levels of MIR106B were increased in intestinal epithelia from patients with active CD, whereas levels of ATG16L1 were reduced compared with controls. Levels of c-Myc were also increased in intestinal epithelia of patients with active CD compared with controls. These alterations could impair removal of CD-associated bacteria by autophagy.

Conclusions

In human cell lines, MIR106B and MIR93 reduce levels of ATG16L1 and autophagy and prevent autophagy-dependent eradication of intracellular bacteria. This process also appears to be altered in colon tissues from patients with active CD.

Section snippets

Cell Lines

Human cell lines HCT116, SW480 (colon cancer), HeLa (cervical cancer), and U2OS (osteosarcoma) were maintained in the laboratory. HCT116/GFP-LC3, SW480/GFP-LC3, and U2OS/GFP-LC3 were parental cell lines stably transfected with GFP-LC3 and established in the laboratory.¹⁸ HCT116 cells were grown in McCoy's 5A medium with 10% fetal bovine serum (FBS). Other cells were cultured in Dulbecco's modified Eagle medium with l-glutamine and 10% FBS. HeLa/mCherry-LC3 cell line was established by stably

Silencing of Dicer Enhances the Expression of ATGs and Formation of Autophagosomes

To determine whether abrogation of miRNA biogenesis is sufficient to induce ATG expression and autophagy, small interfering RNA (siRNA)-mediated knockdown of Dicer was performed.²² We transfected Dicer1 siRNA into U2OS/GFP-LC3 cells, a cell line we previously established for monitoring autophagy.¹⁸ On serum starvation, GFP-LC3 in control cells redistributed from a diffuse cytoplasmic location (Figure 1Ai) to discrete vesicular structures (Figure 1Aii and iii), an indication of autophagy.¹⁸ The

Discussion

Autophagy has been shown to be essential for the clearance of intracellular microbes in mammalian cells.1, 2, 3, 4, 32 However, the underlying mechanism controlling autophagy in these cells remains unknown. We previously reported that the miR-106 cluster was among the miRNAs down-regulated during differentiation of human monocytes into dendritic cells.²³ We also noted that ATG16L1 and ATG9 levels were substantially increased and that autophagy was markedly induced during differentiation of

Acknowledgments

The authors thank Dr Philip M. Sherman (Research Institute of Hospital for Sick Children, Toronto, Ontario, Canada) for providing the AIEC strain LF82, Dr Ramnik Xavier (Harvard University) for providing the original FLAG-ATG16L1 plasmid, and Drs Xuxian Xiao, Weiming Zhang, and Jian Wang in the Xu Laboratory for critical technical support.

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: Author names in bold designate shared co-first authorship

: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by National Cancer Institute grant R01CA133053, the National Cancer Institute Cervical Cancer SPORE Career Development Award and Pilot Award P50CA098252 (to Z.-X.X.), and National Institute of Allergy and Infectious Diseases grant R21AI092220 (to X.Z.).

^∗: Authors share co-first authorship

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Original ResearchFull Report: Basic and Translational—Alimentary TractMIR106B and MIR93 Prevent Removal of Bacteria From Epithelial Cells by Disrupting ATG16L1-Mediated Autophagy

Background & Aims

Methods

Results

Conclusions

Section snippets

Cell Lines

Silencing of Dicer Enhances the Expression of ATGs and Formation of Autophagosomes

Discussion

Acknowledgments

Cell

Cell Host Microbe

Int J Biochem Cell Biol

Blood

Blood

Cell Signal

J Crohns Colitis

Regulation mechanisms and signaling pathways of autophagy

Annu Rev Genet

Autophagy in immunity against intracellular bacteria

Curr Top Microbiol Immunol

A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells

Nature

Autophagy and its role in MHC-mediated antigen presentation

J Immunol

Molecular basis of canonical and bactericidal autophagy

Int Immunol

Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly

Cell Microbiol

Impaired autophagy of an intracellular pathogen induced by a Crohn's disease associated ATG16L1 variant

PLoS One

Epidemiology and natural history of inflammatory bowel diseases

Gastroenterology

Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis

Nat Genet

Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease

Nature

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci

Nat Genet

A microRNA polycistron as a potential human oncogene

Nature

c-Myc-regulated microRNAs modulate E2F1 expression

Nature

Original Research
Full Report: Basic and Translational—Alimentary Tract
MIR106B and MIR93 Prevent Removal of Bacteria From Epithelial Cells by Disrupting ATG16L1-Mediated Autophagy