miR-375 targets 3'-phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells.
OBJECTIVE
MicroRNAs are short, noncoding RNAs that regulate gene expression. We hypothesized that the phosphatidylinositol 3-kinase (PI 3-kinase) cascade known to be important in beta-cell physiology could be regulated by microRNAs. Here, we focused on the pancreas-specific miR-375 as a potential regulator of its predicted target 3'-phosphoinositide-dependent protein kinase-1 (PDK1), and we analyzed its implication in the response of insulin-producing cells to elevation of glucose levels.


METHODS
We used insulinoma-1E cells to analyze the effects of miR-375 on PDK1 protein level and downstream signaling using Western blotting, glucose-induced insulin gene expression using quantitative RT-PCR, and DNA synthesis by measuring thymidine incorporation. Moreover, we analyzed the effect of glucose on miR-375 expression in both INS-1E cells and primary rat islets. Finally, miR-375 expression in isolated islets was analyzed in diabetic Goto-Kakizaki (GK) rats.


RESULTS
We found that miR-375 directly targets PDK1 and reduces its protein level, resulting in decreased glucose-stimulatory action on insulin gene expression and DNA synthesis. Furthermore, glucose leads to a decrease in miR-375 precursor level and a concomitant increase in PDK1 protein. Importantly, regulation of miR-375 expression by glucose occurs in primary rat islets as well. Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets.


CONCLUSIONS
Our findings provide evidence for a role of a pancreatic-specific microRNA, miR-375, in the regulation of PDK1, a key molecule in PI 3-kinase signaling in pancreatic beta-cells. The effects of glucose on miR-375 are compatible with the idea that miR-375 is involved in glucose regulation of insulin gene expression and beta-cell growth.
El Ouaamari
Abdelfattah
A
Institut National de la Santé et de la Recherche Médicale, U907, Nice, France.
Baroukh
Nadine
N
Martens
Geert A
GA
Lebrun
Patricia
P
Pipeleers
Daniel
D
van Obberghen
Emmanuel
E
D020413:3' Untranslated Regions / Q000235:genetics; D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
D020413:3' Untranslated Regions; D007328:Insulin; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578579:Pdpk1 protein, rat; D017346:Protein Serine-Threonine Kinases; D005947:Glucose