Microbial Disruption of Autophagy Alters Expression of the RISC Component AGO2, a Critical Regulator of the miRNA Silencing Pathway.
BACKGROUND
Autophagy is implicated in Crohn's disease (CD) pathogenesis. Recent evidence suggests autophagy regulates the microRNA (miRNA)-induced silencing complex (miRISC). Therefore, autophagy may play a novel role in CD by regulating expression of miRISC, thereby altering miRNA silencing. As microbes associated with CD can alter autophagy, we hypothesized that microbial disruption of autophagy affects the critical miRISC component AGO2.


METHODS
AGO2 expression was assessed in epithelial and immune cells, and intestinal organoids with disrupted autophagy. Microarray technology was used to determine the expression of downstream miRNAs in cells with defective autophagy.


RESULTS
Increased AGO2 was detected in autophagy-deficient ATG5-/- and ATG16-/- mouse embryonic fibroblast cells (MEFs) in comparison with wild-type MEFs. Chemical agents and VacA toxin, which disrupt autophagy, increased AGO2 expression in MEFs, epithelial cells lines, and human monocytes, respectively. Increased AGO2 was also detected in ATG7-/- intestinal organoids, in comparison with wild-type organoids. Five miRNAs were differentially expressed in autophagy-deficient MEFs. Pathway enrichment analysis of the differentially expressed miRNAs implicated signaling pathways previously associated with CD.


CONCLUSIONS
Taken together, our results suggest that autophagy is involved in the regulation of the critical miRISC component AGO2 in epithelial and immune cells and primary intestinal epithelial cells. We propose a mechanism by which autophagy alters miRNA expression, which likely impacts the regulation of CD-associated pathways. Furthermore, as enteric microbial products can manipulate autophagy and AGO2, our findings suggest a novel mechanism by which enteric microbes could influence miRNA to promote disease.
*Cell Biology Program, Hospital for Sick Children, Toronto, ON, Canada; †Departments of Pediatrics and Physiology, University of Toronto, Toronto, ON, Canada; ‡Zane Cohen Centre for Digestive Diseases, Mount Sinai Hospital IBD Group, Toronto, ON, Canada; and §Department of Immunology, University of Toronto, Toronto, ON, Canada.
Michal
M
Sibony
Majd
M
Abdullah
Laura
L
Greenfield
Deepa
D
Raju
Ted
T
Wu
David M
DM
Rodrigues
Esther
E
Galindo-Mata
Heidi
H
Mascarenhas
Dana J
DJ
Philpott
Mark S
MS
Silverberg
Nicola L
NL
Jones
D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000235:genetics*; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D001427:Bacterial Toxins / Q000378:metabolism*; D002352:Carrier Proteins; D002460:Cell Line; D003424:Crohn Disease / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D004847:Epithelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000172:deficiency; D009000:Monocytes / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*
C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; C495773:Atg5 protein, mouse; C502661:Atg7 protein, mouse; D000071187:Autophagy-Related Protein 5; D001427:Bacterial Toxins; D002352:Carrier Proteins; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D034743:RNA-Induced Silencing Complex; D000071193:Autophagy-Related Protein 7