MiR-145 targeting BNIP3 reduces apoptosis of chondrocytes in osteoarthritis through Notch signaling pathway.

W-F Wang, S-Y Liu, Z-F Qi, Z-H Lv, H-R Ding, W-J Zhou

European review for medical and pharmacological sciences 2020 Aug

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The purpose of this study was to explore the effect of micro ribonucleic acid (miR)-145 on the apoptosis of chondrocytes in osteoarthritis (OA), and to research the association between its targeting on B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and Notch signaling pathway and chondrocyte apoptosis. The mouse model of OA was established via surgery, and chondrocytes were isolated and cultured in vitro. Then, the chondrocytes were transfected with miR-145 inhibitor, miR-145 mimics, miR-negative control (NC), BNIP3-siRNA and BNIP3-vector, respectively, with those normally cultured as the control. After that, the expression levels of miR-145 and BNIP3 in cells were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), the apoptosis rate was detected via flow cytometry, and the apoptosis level was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the target gene sequences were predicted and compared using the software, and the BNIP3 Luciferase reporter vectors containing predicted target sites for miR-145 were constructed. Finally, the protein expressions of BNIP3, Notch1, and P21 were determined through Western blotting. The results of qRT-PCR showed that in OA chondrocytes, the expression of miR-145 was lower than that in normal chondrocytes (p<0.05), while the mRNA and protein expressions of BNIP3 were higher than those in normal chondrocytes (p<0.05). According to flow cytometry, the apoptosis rate was (4.4±0.6)% in normal cartilage tissues and (29.2±2.1)% in OA cartilage tissues. Overexpression of miR-145 significantly reduced chondrocyte apoptosis (p<0.05), while overexpression of BNIP3 markedly increased chondrocyte apoptosis (p<0.05). In addition, the Luciferase reporter system showed that miR-145 mimics evidently inhibited BNIP3 (p<0.05) and suppressed the Notch signaling pathway (p<0.05), while BNIP3 enhanced the expression of Notch signaling pathway (p<0.05). MiR-145 can reduce OA-induced chondrocyte apoptosis through targeted inhibition on BNIP3 and regulation on Notch signaling pathway.

Citation

W-F Wang, S-Y Liu, Z-F Qi, Z-H Lv, H-R Ding, W-J Zhou. MiR-145 targeting BNIP3 reduces apoptosis of chondrocytes in osteoarthritis through Notch signaling pathway. European review for medical and pharmacological sciences. 2020 Aug;24(16):8263-8272