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.

  • 1

    Bernardo F , Rebordão L , Machado S , In-hospital and long-term prognosis after spontaneous intracerebral hemorrhage among young adults aged 18-65 years . J Stroke Cerebrovasc Dis . 2019 ;28 (11 ):104350 .

    • Search Google Scholar
    • Export Citation
  • 2

    Wilkinson DA , Pandey AS , Thompson BG , Injury mechanisms in acute intracerebral hemorrhage . Neuropharmacology . 2018 ;134 (Pt B ):240 248 .

    • Search Google Scholar
    • Export Citation
  • 3

    Zhou HJ , Tang T , Cui HJ , Thrombin-triggered angiogenesis in rat brains following experimental intracerebral hemorrhage . J Neurosurg . 2012 ;117 (5 ):920 928 .

    • Search Google Scholar
    • Export Citation
  • 4

    Lan X , Han X , Li Q , Modulators of microglial activation and polarization after intracerebral haemorrhage . Nat Rev Neurol . 2017 ;13 (7 ):420 433 .

    • Search Google Scholar
    • Export Citation
  • 5

    Cui HJ , Yang AL , Zhou HJ , Buyang huanwu decoction promotes angiogenesis via vascular endothelial growth factor receptor-2 activation through the PI3K/Akt pathway in a mouse model of intracerebral hemorrhage . BMC Complement Altern Med . 2015 ;15 :91 .

    • Search Google Scholar
    • Export Citation
  • 6

    Li HT , Zhou HJ , Zhong JH , 2-methoxyestradiol inhibits intracerebral hemorrhage-induced angiogenesis in rats . Turk Neurosurg . 2018 ;28 (2 ):241 247 .

    • Search Google Scholar
    • Export Citation
  • 7

    Hanjin C , Tao L , Pengfei L , Altered long noncoding RNA and messenger RNA expression in experimental intracerebral hemorrhage - a preliminary study . Cell Physiol Biochem . 2018 ;45 (3 ):1284 1301 .

    • Search Google Scholar
    • Export Citation
  • 8

    al Taleb Z , Petry A , Chi TF , Differential transcriptional regulation of hypoxia-inducible factor-1α by arsenite under normoxia and hypoxia: involvement of Nrf2 . J Mol Med (Berl) . 2016 ;94 (10 ):1153 1166 .

    • Search Google Scholar
    • Export Citation
  • 9

    Kuschel A , Simon P , Tug S . Functional regulation of HIF-1α under normoxia—is there more than post-translational regulation? J Cell Physiol . 2012 ;227 (2 ):514 524 .

    • Search Google Scholar
    • Export Citation
  • 10

    Gao F , Zheng M , Hua Y , Acetazolamide attenuates thrombin-induced hydrocephalus . Acta Neurochir Suppl . 2016 ;121 :373 377 .

  • 11

    Yang AL , Zhou HJ , Lin Y , Thrombin promotes the expression of thrombospondin-1 and -2 in a rat model of intracerebral hemorrhage . J Neurol Sci . 2012 ;323 (1-2 ):141 146 .

    • Search Google Scholar
    • Export Citation
  • 12

    Omidkhoda N , Wallace Hayes A , Reiter RJ , Karimi G . The role of microRNAs on endoplasmic reticulum stress in myocardial ischemia and cardiac hypertrophy . Pharmacol Res . 2019 ;150 :104516 .

    • Search Google Scholar
    • Export Citation
  • 13

    Blasiak J , Watala C , Tuuminen R , Expression of VEGFA-regulating miRNAs and mortality in wet AMD . J Cell Mol Med . 2019 ;23 (12 ):8464 8471 .

    • Search Google Scholar
    • Export Citation
  • 14

    Lanigan SM , O’Connor JJ . Prolyl hydroxylase domain inhibitors: can multiple mechanisms be an opportunity for ischemic stroke? Neuropharmacology . 2019 ;148 :117 130 .

    • Search Google Scholar
    • Export Citation
  • 15

    Schreiber T , Salhöfer L , Quinting T , Fandrey J . Things get broken: the hypoxia-inducible factor prolyl hydroxylases in ischemic heart disease . Basic Res Cardiol . 2019 ;114 (3 ):16 .

    • Search Google Scholar
    • Export Citation
  • 16

    Cheng T , Wang W , Li Q , Cerebroprotection of flavanol (-)-epicatechin after traumatic brain injury via Nrf2-dependent and -independent pathways . Free Radic Biol Med . 2016 ;92 :15 28 .

    • Search Google Scholar
    • Export Citation
  • 17

    Kilkenny C , Browne WJ , Cuthill IC , Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research . Osteoarthritis Cartilage . 2012 ;20 (4 ):256 260 .

    • Search Google Scholar
    • Export Citation
  • 18

    Zhou HJ , Yang X , Cui HJ , Leukemia inhibitory factor contributes to reactive astrogliosis via activation of signal transducer and activator of transcription 3 signaling after intracerebral hemorrhage in rats . J Neurotrauma . 2017 ;34 (8 ):1658 1665 .

    • Search Google Scholar
    • Export Citation
  • 19

    Zhou J , Liu T , Guo H , Lactate potentiates angiogenesis and neurogenesis in experimental intracerebral hemorrhage . Exp Mol Med . 2018 ;50 (7 ):78 .

    • Search Google Scholar
    • Export Citation
  • 20

    Ge X , Han Z , Chen F , MiR-21 alleviates secondary blood-brain barrier damage after traumatic brain injury in rats . Brain Res . 2015 ;1603 :150 157 .

    • Search Google Scholar
    • Export Citation
  • 21

    Ge XT , Lei P , Wang HC , miR-21 improves the neurological outcome after traumatic brain injury in rats . Sci Rep . 2014 ;4 :6718 .

  • 22

    Hu E , Hu W , Yang A , Thrombin promotes pericyte coverage by Tie2 activation in a rat model of intracerebral hemorrhage . Brain Res . 2019 ;1708 :58 68 .

    • Search Google Scholar
    • Export Citation
  • 23

    Guo D , Liu J , Wang W , Alteration in abundance and compartmentalization of inflammation-related miRNAs in plasma after intracerebral hemorrhage . Stroke . 2013 ;44 (6 ):1739 1742 .

    • Search Google Scholar
    • Export Citation
  • 24

    Liu DZ , Tian Y , Ander BP , Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures . J Cereb Blood Flow Metab . 2010 ;30 (1 ):92 101 .

    • Search Google Scholar
    • Export Citation
  • 25

    Wu P , Zuo X , Ji A . Stroke-induced microRNAs: the potential therapeutic role for stroke . Exp Ther Med . 2012 ;3 (4 ):571 576 .

    • Search Google Scholar
    • Export Citation
  • 26

    Bang C , Fiedler J , Thum T . Cardiovascular importance of the microRNA-23/27/24 family . Microcirculation . 2012 ;19 (3 ):208 214 .

    • Search Google Scholar
    • Export Citation
  • 27

    Lin SC , Liu CJ , Lin JA , miR-24 up-regulation in oral carcinoma: positive association from clinical and in vitro analysis . Oral Oncol . 2010 ;46 (3 ):204 208 .

    • Search Google Scholar
    • Export Citation
  • 28

    Yin JY , Deng ZQ , Liu FQ , Association between mir-24 and mir-378 in formalin-fixed paraffin-embedded tissues of breast cancer . Int J Clin Exp Pathol . 2014 ;7 (7 ):4261 4267 .

    • Search Google Scholar
    • Export Citation
  • 29

    Zhou J , Zhang J . Identification of miRNA-21 and miRNA-24 in plasma as potential early stage markers of acute cerebral infarction . Mol Med Rep . 2014 ;10 (2 ):971 976 .

    • Search Google Scholar
    • Export Citation
  • 30

    Yang Y , Zhang M , Kang X , Impaired adult hippocampal neurogenesis and cognitive ability in a mouse model of intrastriatal hemorrhage . Neurosci Lett . 2015 ;599 :133 139 .

    • Search Google Scholar
    • Export Citation
  • 31

    Cheng B , Liu F , Guo Q , Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia . PeerJ . 2019 ;7 :e7716 .

    • Search Google Scholar
    • Export Citation
  • 32

    Liao XJ , Mao WM , Wang Q , MicroRNA-24 inhibits serotonin reuptake transporter expression and aggravates irritable bowel syndrome . Biochem Biophys Res Commun . 2016 ;469 (2 ):288 293 .

    • Search Google Scholar
    • Export Citation
  • 33

    Lynch SM , McKenna MM , Walsh CP , McKenna DJ . miR-24 regulates CDKN1B/p27 expression in prostate cancer . Prostate . 2016 ;76 (7 ):637 648 .

    • Search Google Scholar
    • Export Citation
  • 34

    Liu R , Zhang H , Wang X , The miR-24-Bim pathway promotes tumor growth and angiogenesis in pancreatic carcinoma . Oncotarget . 2015 ;6 (41 ):43831 43842 .

    • Search Google Scholar
    • Export Citation
  • 35

    Hägg M , Wennström S . Activation of hypoxia-induced transcription in normoxia . Exp Cell Res . 2005 ;306 (1 ):180 191 .

  • 36

    Bogdanovski DA , DiFazio LT , Bogdanovski AK , Hypoxia-inducible-factor-1 in trauma and critical care . J Crit Care . 2017 ;42 :207 212 .

    • Search Google Scholar
    • Export Citation
  • 37

    Du Y , Ge Y , Xu Z , Hypoxia-inducible factor 1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF) pathway participates in angiogenesis of myocardial infarction in muscone-treated mice: preliminary study . Med Sci Monit . 2018 ;24 :8870 8877 .

    • Search Google Scholar
    • Export Citation
  • 38

    Yang X , Tang X , Sun P , MicroRNA-15a/16-1 antagomir ameliorates ischemic brain injury in experimental stroke . Stroke . 2017 ;48 (7 ):1941 1947 .

    • Search Google Scholar
    • Export Citation
  • 39

    Bai R , Gao H , Han Z , Long-term kinetics of immunologic components and neurological deficits in rats following repetitive mild traumatic brain injury . Med Sci Monit . 2017 ;23 :1707 1718 .

    • Search Google Scholar
    • Export Citation
  • 40

    Horiquini Barbosa E , Vallim JH , Lachat JJ , de Castro VL . Assessments of motor abnormalities on the grid-walking and foot-fault tests from undernutrition in Wistar rats . J Mot Behav . 2016 ;48 (1 ):5 12 .

    • Search Google Scholar
    • Export Citation
  • 41

    McBride DW , Wang Y , Adam L , Correlation between subacute sensorimotor deficits and brain edema in rats after surgical brain injury . Acta Neurochir Suppl . 2016 ;121 :317 321 .

    • Search Google Scholar
    • Export Citation
  • 42

    Lee HJ , Kim KS , Park IH , Kim SU . Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model . PLoS One . 2007 ;2 (1 ):e156 .

    • Search Google Scholar
    • Export Citation
  • 43

    Zhang ZG , Chopp M . Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic . Lancet Neurol . 2009 ;8 (5 ):491 500 .

    • Search Google Scholar
    • Export Citation
  • 44

    Pan C , Liu N , Zhang P , EGb761 ameliorates neuronal apoptosis and promotes angiogenesis in experimental intracerebral hemorrhage via RSK1/GSK3β pathway . Mol Neurobiol . 2018 ;55 (2 ):1556 1567 .

    • Search Google Scholar
    • Export Citation
  • 45

    Chen N , Huang CX , Huang CH , The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1 . Gene . 2018 ;678 :219 225 .

    • Search Google Scholar
    • Export Citation

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