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Published on: June 9, 2018
TLR4 knockdown by miRNA-140-5p improves tendinopathy: an in vitro study
Tu Jun1, Guo Ruipeng1, Xu Bin1
1Department of Sports Medicine and Arthroscopic Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei City, Anhui Province, China.
Introduction:
The purpose of this study was to determine whether TLR4 knockdown induced by miRNA-140-5p improves tendinopathy in an in vitro experiment.
Material And Methods:
Extraction of tendon-derived stem cells (TDSCs) from SD rats was performed using TGF-β1 to develop a tendinopathy cell model. In the first step, we knocked down TLR4 by si-TLR4 to investigate TLR4 in tendinopathy development, and the next we used miRNA-140-5p to investigate miRNA-140-5p in tendinopathy development. The inflammatory factors and Hyp concentration were evaluated by ELISA assay; the cell viability was measured by MTT assay; the cell apoptosis was evaluated by TUNEL and/or flow cytometry. The relative mRNA was measured by RT-qPCR assay; the relative proteins expression was evaluated by cellular immunofluorescence and/or WB assay. The correlation between miRNA-140-5p and TLR4 was analyzed by Luciferase reporter assay.
Results:
With miRNA-140-5p overexpression or TLR4 knockdown, the cell viability was significantly increased with cell apoptosis depressing compared with the Model group (p < 0.05, respectively). Meanwhile, the inflammatory factors TNF-α, IL-1β and IL-6 and Hyp concentration were significantly improved (p < 0.05, respectively), whereas the TLR4, MyD88 and NF-κB(p65) protein expression levels were significantly depressed with TLR4 knockdown by si-TLR4 or miRNA-140-5p which target TLR4.
Conclusions:
The present results showed that TLR4 knockdown induced by miRNA-140-5p or si-TLR4 improved tendinopathy in an in vitro cell experiment.
