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

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.

Keywords: CeRNA; Endothelial cells; Enoxaparin sodium; High-throughput RNA sequencing; Thrombosis.

Fig. 1

a High-viscosity bone cement powder and enoxaparin sodium (ES) samples. b The extracorporeal shunt and prepared silk thread with bone cement. c The exposed arteriovenous vessels of New Zealand rabbits. d Establishment of the animal model. e Blood vessels contacting the thread covered with bone cement. f Intravascular bone cement-induced thrombus samples

Fig. 2

a PMMA. There were many gaps between the bone cement particles. b ES-PMMA. The surface of the bone cement particles was covered with a syrup-like substance, which was considered the attached enoxaparin

Fig. 3

The mRNA expression levels of CD31, CD40, CD62p, CD106, endothelin, and thrombomodulin showed that CD31 and CD40 expression was decreased in the M group compared with the Con group (P < 0.01) and that CD40 mRNA expression was significantly decreased in the M group compared with the Con group (P < 0.001)

Fig. 4

Protein expression levels of CD31, CD40, CD62p, CD106, endothelin, and thrombomodulin. The expression levels of CD31 and CD40 were decreased in the M group compared with the Con group (P < 0.05), and the expression levels of CD40 were significantly decreased in the M group compared with the Con group (P < 0.01)

Fig. 5

The average Q values of single individual box plots

Fig.6

Distribution of sample sequence coverage

Fig. 7

a: Heat map of differentially expressed total lncRNAs, b Heat map of newly discovered lncRNAs

Fig. 8

CD40 gene with the ceRNA hypothesis

Fig. 9

Low lncRNA MSTRG.22719.16 fluorescent probe expression in the M group in the figure

Fig. 10

A Schematic of the ocu-miR-326-5p binding site in the 3′UTR of CD40 mRNA and the corresponding mutation. B Schematic of the ocu-miR-326-5p binding site in the partial base sequence of lncRNA MSTRG.22719.16 and the corresponding mutation. C pmirGLO-CD40 (Wt)/(Mut), pmirGLO-lncRNAMSTRG.22719.16(Wt)/(Mut) and negative control (NC), ocu-miR-326-5p mimics, inhibitor negative control (INC), and ocu-miR-326-5p mimic inhibitor were cotransfected into 293 T cells, and luciferase activity was determined, *P < 0.05, **P < 0.01. The results showed that the 3′UTR of CD40 mRNA and the base sequence of lncRNA MSTRG.22719.16 were able to bind to ocu-miR-326-5p mimics