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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.

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miR-200c was upregulated in the degenerative NP tissues and involved in the regulation of NPC viability and functions. (A) Differential upregulation of miRNAs detected by microarray in degenerative NP tissues compared with controls. Volcano plots were constructed using fold-change values and P values. The vertical green line corresponds to 2.0-fold upregulation between degenerative samples and controls, and the horizontal green line represents a P value of 0.01. The red point in the plot represents the differentially upregulated miRNAs with statistical significance. (B) qRT-PCR analysis confirmed the upregulated miRNAs in the degenerative NP samples from patients with IVDD. n=12; **P<0.01 compared with the controls. (C) qRT-PCR analysis confirmed the upregulated miRNAs in the degenerative NP samples from the rat model of IVDD. n=8; **P<0.01 compared with the controls. (D) Representative northern blots showing miR-200c levels in the NP samples from patients with or without IVDD. (E) NPCs were transfected with miR-200c, miR-negative control (NC), antagomir-200c or antagomir-NC. miR-200c levels in NPCs were analysed by qRT-PCR. **P<0.01. (F) Representative dot plots of apoptosis flow cytometry detection were shown after Annexin V-FITC/propidium iodide (PI) dual staining. The transfection of miR-200c increased apoptosis rate of 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 (ECM) compositions (collagen II, aggrecan) in NPCs after transfection of miR-200c. (H) NPCs were transfected with miR-200c antagonist or its NC, and then treated with inflammatory cytokines (IC; interleukin 1β plus tumour necrosis factor α). qRT-PCR showed increased miR-200c levels in NPCs treated with IC, which could be converted by transfection of miR-200c antagonist. **P<0.01. (I) Representative dot plots of apoptosis flow cytometry detection were shown after Annexin V-FITC/PI dual staining. The knockdown of miR-200c inhibited apoptosis induced by IC in NPCs. **P<0.01. (J) Western blot analysis showed that miR-200c knockdown attenuated the apoptotic and catabolic response and reversed the decreased expression of ECM compositions induced by IC treatment in NPCs. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IVDD, intervertebral disc degeneration; miRNA, microRNAs; MMP, matrix metalloproteinases; NP, nucleus pulposus; NPC, nucleus pulposus cells; qRT-PCR, quantitative real-time reverse transcription-PCR.