LncRNA MSTRG.22719.16 通过靶向 ocu-miR-326-5p/CD40 轴介导依诺肝素钠高黏度骨水泥诱导血栓形成的减少。

LncRNA MSTRG.22719.16 mediates the reduction of enoxaparin sodium high-viscosity bone cement-induced thrombosis by targeting the ocu-miR-326-5p/CD40 axis.

机构信息

Department of Orthopaedic Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, China.

Department of Orthopaedic Surgery, Faculty of Medicine, Oita University, Oita, Japan.

出版信息

J Orthop Surg Res. 2023 Sep 22;18(1):716. doi: 10.1186/s13018-023-04109-5.

DOI:10.1186/s13018-023-04109-5

PMID:37736740

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10514947/

Abstract

OBJECTIVE

Polymethylmethacrylate (PMMA) bone cement promotes the development of local thrombi. Our study found that a novel material, ES-PMMA bone cement, can reduce local thrombosis. We used a simple and reproducible animal model to confirm the reduction in local thrombosis and explored the associated molecular mechanism.

METHODS

New Zealand rabbits, which were used to model thrombosis using extracorporeal carotid artery shunts, were divided into the following two groups, with 3 rabbits in each group: the PMMA bone cement group and the ES-PMMA bone cement group. Four hours after modelling, experimental samples, including thrombotic and vascular tissues, were collected. Thrombotic samples from the PMMA group and ES-PMMA group were subjected to lncRNA sequencing, and a lncRNA microarray was used to screen the differentially expressed lncRNAs. The expression of thrombomodulin in endothelial cells was quantified in vascular tissue samples. Differences in the lncRNA expression profiles between the thrombotic samples of the PMMA group and ES-PMMA group were assessed by base-to-base alignment in the intergenic regions of genomes. The lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network was established in light of ceRNA theory. Thrombosis was observed in the PMMA group and ES-PMMA group.

RESULTS

The thrombotic weight was 0.00706 ± 0.00136 g/cm in the PMMA group and 0.00551 ± 0.00115 g/cm in the ES-PMMA group. Quantitative real-time polymerase chain reaction (RT-q-CR) and Western blotting revealed that the expression of CD40, which can regulate thrombosis in vascular endothelial cells, was significantly lower in the ES-PMMA group than in the PMMA group. High-throughput sequencing was used to identify 111 lncRNAs with lower expression in the ES-PMMA group than in the PMMA group. Through bioinformatics investigation, lncRNA MSTRG22719.16/ocu-miR-326-5p/CD40 binding sites were selected. Fluorescent in situ RNA hybridization (FISH) was performed to verify the lower expression of lncRNA MSTRG.22719.16 in vascular tissues from the ES-PMMA group. A dual-luciferase reporter gene assay was applied to verify that ocu-miR-326-5p binds the CD40 3'-UTR and targets lncRNA MSTRG.22719.16.

CONCLUSION

Compared with PMMA bone cement, ES-PMMA bone cement can reduce thrombosis through the lncRNA MSTRG.22719.16/ocu-miR-326-5p/CD40 axis.

摘要

目的

聚甲基丙烯酸甲酯（PMMA）骨水泥可促进局部血栓形成。我们的研究发现，一种新型材料 ES-PMMA 骨水泥可减少局部血栓形成。我们使用一种简单且可重复的动物模型证实了局部血栓形成的减少，并探讨了相关的分子机制。

方法

采用新西兰兔建立体外颈动脉分流模型，将其分为以下两组，每组 3 只：PMMA 骨水泥组和 ES-PMMA 骨水泥组。建模 4 小时后，采集血栓和血管组织等实验样本。PMMA 组和 ES-PMMA 组的血栓样本进行长链非编码 RNA 测序，采用长链非编码 RNA 芯片筛选差异表达的长链非编码 RNA。对血管组织样本中血管内皮细胞中血栓调节蛋白的表达进行定量。PMMA 组和 ES-PMMA 组血栓样本的长链非编码 RNA 表达谱通过基因组间区的碱基比对进行评估。根据 ceRNA 理论建立长链非编码 RNA-miRNA-mRNA 竞争内源性 RNA（ceRNA）网络。观察 PMMA 组和 ES-PMMA 组的血栓形成情况。

结果

PMMA 组血栓重量为 0.00706±0.00136 g/cm，ES-PMMA 组为 0.00551±0.00115 g/cm。定量实时聚合酶链反应（RT-q-CR）和 Western blot 显示，ES-PMMA 组血管内皮细胞中调节血栓形成的 CD40 表达明显低于 PMMA 组。采用高通量测序技术鉴定出 ES-PMMA 组较 PMMA 组表达下调的长链非编码 RNA 共 111 个。通过生物信息学研究，选择了长链非编码 RNA MSTRG22719.16/ocu-miR-326-5p/CD40 结合位点。采用荧光原位 RNA 杂交（FISH）验证 ES-PMMA 组血管组织中长链非编码 RNA MSTRG.22719.16 表达下调。双荧光素酶报告基因检测验证 ocu-miR-326-5p 结合 CD40 3'-UTR 并靶向长链非编码 RNA MSTRG.22719.16。