miR-145 通过靶向 BNIP3 减少骨关节炎软骨细胞凋亡，该过程通过 Notch 信号通路实现。
MiR-145 targeting BNIP3 reduces apoptosis of chondrocytes in osteoarthritis through Notch signaling pathway.
机构信息
Department of Orthopedic Surgery, Liaocheng People's Hospital, Liaocheng, China.
出版信息
Eur Rev Med Pharmacol Sci. 2020 Aug;24(16):8263-8272. doi: 10.26355/eurrev_202008_22622.
OBJECTIVE
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.
MATERIALS AND METHODS
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.
RESULTS
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).
CONCLUSIONS
MiR-145 can reduce OA-induced chondrocyte apoptosis through targeted inhibition on BNIP3 and regulation on Notch signaling pathway.
目的
本研究旨在探讨微小 RNA（miR）-145 对骨关节炎（OA）软骨细胞凋亡的影响，并研究其对 B 细胞淋巴瘤-2（Bcl-2）/腺病毒 E1B 19 kDa 相互作用蛋白 3（BNIP3）的靶向作用与 Notch 信号通路和软骨细胞凋亡之间的关系。
材料与方法
通过手术建立小鼠 OA 模型，并在体外分离和培养软骨细胞。然后，分别用 miR-145 抑制剂、miR-145 模拟物、miR 阴性对照（NC）、BNIP3-siRNA 和 BNIP3 载体转染软骨细胞，以正常培养的细胞作为对照。然后，通过定量逆转录聚合酶链反应（qRT-PCR）检测细胞中 miR-145 和 BNIP3 的表达水平，通过流式细胞术检测细胞凋亡率，通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）法检测细胞凋亡水平。此外，使用软件预测和比较靶基因序列，并构建含有 miR-145 预测靶位点的 BNIP3 荧光素酶报告载体。最后，通过 Western blot 测定 BNIP3、Notch1 和 P21 的蛋白表达。
结果
qRT-PCR 结果显示，在 OA 软骨细胞中，miR-145 的表达低于正常软骨细胞（p<0.05），而 BNIP3 的 mRNA 和蛋白表达高于正常软骨细胞（p<0.05）。根据流式细胞术，正常软骨组织的细胞凋亡率为（4.4±0.6）%，OA 软骨组织的细胞凋亡率为（29.2±2.1）%。miR-145 的过表达显著降低软骨细胞凋亡（p<0.05），而 BNIP3 的过表达显著增加软骨细胞凋亡（p<0.05）。此外，荧光素酶报告系统显示，miR-145 模拟物明显抑制 BNIP3（p<0.05）并抑制 Notch 信号通路（p<0.05），而 BNIP3 增强 Notch 信号通路的表达（p<0.05）。
结论
miR-145 可通过靶向抑制 BNIP3 和调节 Notch 信号通路来减少 OA 诱导的软骨细胞凋亡。
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