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. 2018 Jan 17;77(5):770–779. doi: 10.1136/annrheumdis-2017-212056

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© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

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miR-200c-regulated NPC viability and functions through inhibiting its target mRNA, X chromosome-linked inhibitor-of-apoptosis protein (XIAP). (A) 3′-UTR region of XIAP mRNA was found to harbour a binding site for miR-200c. (B) NPCs were transfected with miR-200c or miR-negative control (NC), and then transfected with the luciferase constructs of the wild-type XIAP-3′UTR (3′UTR-wt) or the mutated XIAP-3′UTR (3′UTR-mut). Luciferase reporter assay found that miR-200c exclusively decreased luciferase activity of the wild-type reporter plasmids. n=6; **P<0.01. (C) Western blot analysis revealed lower expression of XIAP in the degenerative NP tissues compared with the controls. (D) NPCs were transfected with miR-200c, miR-NC, antagomir-200c or antagomir-NC. Western blot analysis showed that the expression of XIAP was suppressed by miR-200c upregulation and elevated by miR-200c knockdown. (E) NPCs were transfected with antagomir-200c or its NC, and then treated with inflammatory cytokines (IC; interleukin 1β and tumour necrosis factor-α). Western blot analysis showed decreased XIAP expression in NPCs treated with IC, which could be alleviated by transfection of miR-200c antagonist. (F, G) NPCs were cotransfected with antagomir-200c and XIAP siRNA (XIAP-si) or scramble siRNA (scramble-si), and then exposed to IC. (F) Representative dot plots of apoptosis flow cytometry detection were shown after Annexin V-FITC/propidium iodide (PI) dual staining. The knockdown of XIAP attenuated the inhibitory effects of miR-200c antagonist on apoptosis induced by IC in NPCs. **P<0.01. (G) Western blot analysed protein expression of apoptotic effector caspases (caspase-3, caspase-7 and caspase-9), catabolic enzymes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5) and extracellular matrix compositions (collagen II, aggrecan) in NPCs. The knockdown of XIAP interfered with the effects of miR-200c antagonist on the expression of these functional proteins in IC-treated NPCs. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IVDD, intervertebral disc degeneration; MMP, matrix metalloproteinases; NP, nucleus pulposus; NPC, nucleus pulposus cell; UTR, untranslated region.