Thrombin-induced miRNA-24–1-5p upregulation promotes angiogenesis by targeting prolyl hydroxylase domain 1 in intracerebral hemorrhagic rats

Hanjin Cui MD, PhD 1 , Ali Yang MD, PhD 2 , Huajun Zhou MD, PhD 3 , Yang Wang MD, PhD 1 , Jiekun Luo MD, PhD 1 , Jun Zhou MD 4 , Tao Liu MD, PhD 1 , Pengfei Li MD, PhD 1 , Jing Zhou MD, PhD 1 , En Hu MD 1 , Zehui He MD 1 , Wang Hu MD 1 and Tao Tang MD, PhD 1
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  • 1 Institute of Integrative Medicine and
  • 4 Institute of Medical Science, Xiangya Hospital, Central South University, Changsha, Hunan;
  • 2 Department of Neurology, Henan Province People’s Hospital, Zhengzhou; and
  • 3 Institute of Neurology, The First College of Clinical Medical Sciences, China Three Gorges University, Yichang, Hubei, China
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OBJECTIVE

Thrombin is a unique factor that triggers post-intracerebral hemorrhage (ICH) angiogenesis by increasing hypoxia-inducible factor–1α (HIF-1α) at the protein level. However, HIF-1α mRNA remains unchanged. MicroRNAs (miRNAs) mediate posttranscriptional regulation by suppressing protein translation from mRNAs. This study aimed to determine if miRNAs might be involved in thrombin-induced angiogenesis after ICH by targeting HIF-1α or its upstream prolyl hydroxylase domains (PHDs).

METHODS

The study was divided into two parts. In part 1, rats received an injection of thrombin into the right globus pallidus. An miRNA array combined with miRNA target prediction, luciferase activity assay, and miRNA mimic/inhibitor transfection were used to identify candidate miRNAs and target genes. Part 2 included experiments 1 and 2. In experiment 1, rats were randomly divided into the sham group, ICH group, and ICH+hirudin–treated (thrombin inhibitor) group. In experiment 2, the rats were randomly divided into the sham group, ICH group, ICH+antagomir group, ICH+antagomir-control group, and ICH+vehicle group. Western blotting and quantitative real-time polymerase chain reaction were used to determine the expression of protein and miRNA, respectively. The coexpression of miR-24–1-5p (abbreviated to miR-24) and von Willebrand factor was detected by in situ hybridization and immunohistochemical analysis. The angiogenesis was evaluated by double-labeling immunofluorescence. Neurological function was evaluated by body weight, modified Neurological Severity Scores, and corner turn and foot-fault tests.

RESULTS

In part 1, it was shown that miR-24, which is predicted to target PHD1, was upregulated (fold-change of 1.83) after thrombin infusion, and that the miR-24 mimic transfection decreased luciferase activity and downregulated PHD1 expression (p < 0.05). miR-24 inhibitor transfection increased PHD1 expression (p < 0.05). In part 2, it was shown that miR-24 was expressed in endothelial cells. The HIF-1α protein level and proliferating cell nuclear antigen–positive (PCNA+) nuclei in vessels were increased, while the PHD1 protein level was decreased after ICH, and these effects were reversed by hirudin (p < 0.05). The antagomiR-24–treated rats exhibited a markedly lower body weight and significantly poorer recovery from neurological deficit compared with those in ICH groups (p < 0.05). AntagomiR-24 intervention also led to lower miR-24 expression, a higher PHD1 protein level, and fewer PCNA+ nuclei in vessels compared with those in ICH groups (p < 0.05).

CONCLUSIONS

The present study suggests that thrombin reduces HIF-1α degradation and initiates angiogenesis by increasing miR-24, which targets PHD1 after ICH.

ABBREVIATIONS BMEC = brain microvascular endothelial cell; CTT = corner turn test; EC = endothelial cell; FFT = foot-fault test; HIF = hypoxia-inducible factor; ICH = intracerebral hemorrhage; IHC = immunohistochemistry; ISH = in situ hybridization; LCM = laser capture microdissection; miRNA = microRNA; mNSS = modified Neurological Severity Score; PCNA = proliferating cell nuclear antigen; PHD = prolyl hydroxylase domain; qRT-PCR = quantitative real-time polymerase chain reaction; vWF = von Willebrand factor.

Supplementary Materials

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Contributor Notes

Correspondence Tao Tang: Institute of Integrative Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China. tangtaotay@csu.edu.cn.

INCLUDE WHEN CITING Published online May 15, 2020; DOI: 10.3171/2020.2.JNS193069.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

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