Posted on May 12, 2021
Supplementary MaterialsDocument S1
Supplementary MaterialsDocument S1. that this inhibitions of miR-221/222 increased the expression of ATG12 and p27 and functionally induced extended autophagy and cell death of MM cells. In conclusion, our findings demonstrated the crucial role of the miR-221/222-ATG12/p27-mTOR autophagy-regulatory axis in Dex resistance of MM, and they suggest potential prediction and treatment?strategies for glucocorticoid resistance. and and findings, Dex markedly decreased the expressions of miR-221/222 and p62, and it increased the expressions of ATG12 and p27 in MM.1S-xenografted mice, but not in MM.1R-xenografted mice (Figures 5IC5M). Based on these findings, we concluded that Dex-induced miR-221/222 reduction may C1qtnf5 contribute to the occurrence of pro-death autophagy in MM. The Inhibition of miR-221/222 Improves the Autophagy and Dex Sensitivity of MM Cells findings, we observed increased expression of both ATG12 and p27, as well as increased LC3B-II, decreased p62, and increased Beclin-1 in excised tumors treated with antagomir-221/222 (Physique?6B). Moreover, combination treatment with antagomir-221/222 plus Dex induced further upregulation of both ATG12 and p27, as well as extended autophagy in tumor tissues (Figures 6B and 6C). (+)-CBI-CDPI1 Taken together, these data further indicated that miR-221/222 could inhibit the autophagy pathway in MM cells and targeting miR-221/222 could sensitize MM cells to Dex treatment. Open in a separate window Physique?6 Inhibition of miR-221/222 Promotes Autophagy and Restores Dex Sensitivity of MM Cells Luciferase Reporter Assay HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with 2?g plasmids expressing wild-type Luc-ATG12 or mutant Luc-ATG12 (GeneChem, Shanghai, China); 0.4?g vacant plasmids; plasmids expressing miR-221, miR-222, or miR-NC; and 0.02?g Renilla construct in 24-well plates. At 48?h after transfection, cell extracts were prepared, and luciferase reporter assays were performed using the Dual-Luciferase Assay Kit (Promega, Madison, WI, USA). Firefly Luciferase activities were normalized to parallel Renilla activities. Cell Viability Assay Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell viability, according to the manufacturers instructions (Dojindo Laboratories, Kumamoto, Japan). For combination experiments with microRNAs, 8? 105 MM.1S, U266, or JJN3 cells were transfected with agomir-221/222 or agomir-NC (NC) and MM.1R, ARH-77, or NCI-H929 cells were transfected with antagomir-221/222 or antagomir-NC (NC) in 6-well plates. After 24 h, MM cells were re-seeded in 96-well plates (3? 104 cells/well) and treated with Dex (Sigma-Aldrich, St. Louis, MO, USA) at the indicated concentrations for 48 h. For the combination of autophagy inhibitors with Dex experiments, MM cells were seeded in 96-well plates (3? 104 cells/well), pretreated with autophagy inhibitor 3-MA (500?M, Sigma-Aldrich) or Ly294002 (2.5?M, Sigma-Aldrich) for 2 h, followed by Dex (1?M) for 48 h, and then subjected to CCK-8 assay. Similar conditions were performed for combination experiments with siRNAs. Transmission Electron Microscopy Cells were seeded and subjected to Dex treatment in 6-well plates. After 24 (+)-CBI-CDPI1 h, cells were collected and washed twice with PBS. Then, cell pellets were fixed with 2.5% phosphate-buffered glutaraldehyde and stored at 4C before embedding. After washing with PBS, the cells were postfixed with 1% OsO4 (Servicebio, Wuhan, China), dehydrated with an increasing gradient of ethanol and acetone, and then embedded in Spurrs resin. Ultrathin sections (50C70?nm) were obtained on an electron microscope (EM) UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and adhered to uncoated copper grids. The sections were then stained with 4% uranyl acetate and lead citrate prior to viewing on a Tecnai G2 12 transmission electron microscope (FEI, Hillsboro, OR, USA). GFP-mCherry-LC3B Transfection and Confocal Microscopy MM cells were infected with adenovirus harboring vector expressing GFP-mcherry-LC3B fusion protein, according to the manufacturers instructions (Vigenebio, Jinan, China). After the induction of autophagy, MM cells were collected and seeded on glass slides (+)-CBI-CDPI1 coated with polylysine (Servicebio) for 30?min at room heat. Cells then were fixed with 4% paraformaldehyde for 30?min and washed 3 times with PBS for 5?min/wash. The fixed cells were counterstained with DAPI (Antgene, Wuhan, China) for nuclear staining for 15?min, washed three times with PBS, and examined using a Nikon Eclipse Ti laser-scanning confocal microscope (Nikon, Japan)..