S. D'Adamo, O. Alvarez-Garcia, Y. Muramatsu, F. Flamigni, M.K. Lotz

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MicroRNA-155 suppresses autophagy in chondrocytes by modulating expression of autophagy proteins S. D'Adamo, O. Alvarez-Garcia, Y. Muramatsu, F. Flamigni, M.K. Lotz Osteoarthritis and Cartilage Volume 24, Issue 6, Pages 1082-1091 (June 2016) DOI: 10.1016/j.joca.2016.01.005 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 MiR-155 modulates autophagy in T/C28a2 cells. (A) T/C28a2 cells were transfected for 24 h with LNA miR-155 and LNA nc (40 nM), mimic miR-155 and mimic nc (10 nM). MiR-155 levels were determined by qPCR (n = 4 independent experiments). (B) 24 h after transfection cells were treated with DMSO or 50 nM RAPA and/or 25 μM CQ for 4 h or (C) 5 mM 2-DG and/or 25 μM CQ for 2 h and then harvested for LC3 and β-ACTIN detection by western blotting. Representative images and relative quantification for LC3-II/LC3-I ratios are shown (n = 5 independent experiments), (D) 24 h after transfection cells were incubated for 18 h with DMSO or 50 nM RAPA and/or 25 μM CQ or (E) 5 mM 2-DG and/or 25 μM CQ and stained with Cyto-ID (1:1000) and Hoechst 33,342 (1:2000) (n = 3 independent experiments). Values are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 MiR-155 modulates autophagy in human primary chondrocytes. (A) Cells were transfected for 24 h with LNA miR-155 and LNA nc, mimic miR-155 and mimic nc. MiR-155 levels were determined by qPCR (n = 4 independent experiments with separate chondrocyte preparations). (B) 24 h after transfection cells were treated with or without 25 μM CQ for 12 h or (C) DMSO or 50 nM RAPA and/or 25 μM CQ for 12 h and then harvested for LC3 and β-ACTIN detection by western blotting. Representative images and relative quantification for LC3-II/LC3-I ratios are shown (n = 4 independent experiments with separate chondrocyte preparations), (D) 24 h after transfection cells were incubated for 18 h with or without 25 μM CQ or (E) DMSO or 50 nM RAPA and/or 25 μM CQ and stained with Cyto-ID (1:1000) and Hoechst 33,342 (1:2000) and intracellular fluorescence was measured (n = 4 independent experiments with separate chondrocyte preparations). Values are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 MiR-155 regulates autophagy by suppressing gene expression of MAP1LC3, GABARAPL1, ATG3, ATG5, ULK1, ATG14, and FOXO3. T/C28a2 cells were transfected for 24 h with LNA miR-155 and LNA nc, mimic miR-155 and mimic nc. qRT-PCR analysis of mRNA expression levels of direct and indirect targets of miR-155 in T/C28a2 cells following transfection with LNA (A) or (B) mimic (n = 4 independent experiments). Values are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 MiR-155 regulates autophagy by reducing protein levels of MAP1LC3, GABARAPL1, ATG3, ATG5, ULK1, ATG14, and FOXO3. T/C28a2 cells were transfected for 24 h with LNA miR-155 and LNA nc, mimic miR-155 and mimic nc. Western blotting analysis of MAP1LC3, GABARAPL1, ATG3, ATG5, ULK1, ATG14, FOXO3 and β-ACTIN proteins. Representative images (A) and relative quantifications (B) are shown (n = 4 independent experiments). Values are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of miR-155 on mTOR activity. T/C28a2 cells were transfected for 24 h with LNA miR-155 and LNA nc, mimic miR-155 and mimic nc. (A) Western blotting analysis of P-AKT (Ser473), AKT total, P-S6 (Ser235/Ser236), S6 total and β-ACTIN levels by using specific antibodies. (B) Western blotting analysis of RICTOR and β-ACTIN. Representative images and relative quantifications are shown (n = 4 independent experiments). (C) qRT-PCR analysis of mRNA expression levels of RICTOR are shown (n = 4 independent experiments). Values are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Proposed model of miR-155-mediated suppression of autophagy. MiR-155 targets the expression of several autophagy-related key factors: ULK1, involved in the initiation phase; ATG14, belonging to the BECLIN1 complex responsible of the phagophore nucleation; ATG5, ATG3, LC3 and GABARAPL1, crucial components in the elongation and maturation steps; FOXO3 suggested as critical transcription factor of autophagy-related genes, including MAP1LC3, ULK1, ATG14 and GABARAPL1. Even though miR-155-mediated suppression of RICTOR expression led to a reduction of mTOR activity this does not reverse the inhibition of autophagy. Red colors represent autophagy-reducing effects and, on the other side, blue colors are used for autophagy-promoting effects and dashed lines indicate no predicted relationships. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Suppl. Fig. 1 MiR-155 suppresses autophagy-related genes in primary human chondrocytes. Cells were transfected for 24 h with LNA miR-155 and LNA nc, mimic miR-155 and mimic nc. (A) qRT-PCR analysis of mRNA expression levels of direct and indirect targets of miR-155 in human primary chondrocytes following transfection with LNA or (B) mimic (n = 4 independent experiments with separate chondrocyte preparations). Values are expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Osteoarthritis and Cartilage 2016 24, 1082-1091DOI: (10.1016/j.joca.2016.01.005) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions