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. 2016 Sep 15;66(1):177–192. doi: 10.2337/db16-0052

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© 2017 by the American Diabetes Association.

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IRE1α deletion results in the elevation of a subset of miRs. Total RNA was extracted from db/db and db/+ BMPCs for miR microarray analysis. A: Heat map of miR microarray analysis in db/+ and db/db BMPCs. n = 3 per group. B: qRT-PCR analysis of mature miRs of miR-466 and miR-200 families as well as miR-223, miR-155, and miR-34a in db/+ and db/db BMPCs. n = 7 per group. *P < 0.05 vs. db/+. IRE1α^−/− BMPCs were generated by infecting IRE1α^flox/flox BMPCs with Ad-CMV-Cre (100 MOI, Ad-β-Gal infection as control IRE1^+/+ BMPCs). IRE1α protein expression was determined by Western blot analyses. C: IRE1α protein expression in IRE1^+/+ and IRE1^−/− BMPCs. n = 6 per group. D: qRT-PCR analysis of XBP1 mRNA splicing in IRE1α^−/− and IRE1α^+/+ BMPCs. n = 6 per group. *P < 0.05 vs. IRE1α^+/+ BMPCs. E: qRT-PCR analysis of mature miRs in IRE1^−/− and IRE1^+/+ BMPCs. n = 6 per group. The elevation of all the miRs listed in this panel was statistically significant compared to IRE1α^+/+ BMPCs (P < 0.05). F: Upper left illustration of IRE1α-mediated miR decay by destroying pre-miR stem-loop structure. Upper right sequence motif and stem-loop structure for the IRE1α cleavage sites within human XBP1 mRNA, pre-miR-466, and pre-miR-200. Lower pictures show the predicted miR secondary structures for miR-466 and miR-200 with their potential IRE1α cleavage sites (G/C sites marked with red arrows). G: Mature miR expression after pre-miR-466 transfection in either IRE1α^+/+ BMPCs, IRE1^−/− BMPCs, or IRE1α^−/− BMPCs infected with Ad-K907A. Plasmid expressing pre-miR-466 or empty plasmid was transfected into either IRE1α^−/− or IRE1α^+/+ BMPCs via TurboFECT at the concentration of 100 ng/10⁶ cells. miR-466h was detected using real-time PCRs. n = 6 per group. H: Mature miR expression after pre-miR-466 transfection in IRE1α ^−/− BMPCs. Plasmid carrying pre-miR-200 or empty plasmid was transfected into either IRE1α ^+/+ BMPCs, IRE1α ^−/− BMPCs, or IRE1α ^−/− BMPCs infected with Ad-K907A, via TurboFECT at the concentration of 100 ng/10⁶ cells. Levels of miR-200b were analyzed using qRT-PCR. n = 6 per group. I: In vitro IRE1α-mediated pre-miR cleavage assay. In vitro–transcribed pre-miR-200 or pre-miR-466 RNA was incubated with or without recombinant IRE1α protein in the presence or absence of ATP in the reaction buffer. The cleavage reaction products were resolved on an agarose gel. The reaction without ATP initiation serves as a control for each sample. VH, vehicle buffer that did not contain IRE1α protein.