Long non-coding RNA H19 promotes leukocyte inflammation in ischemic stroke by targeting the miR-29b/C1QTNF6 axis

Guangwen Li, Xiaoqing Ma, Haiping Zhao, Junfen Fan, Tianwei Liu, Yumin Luo, Yunliang Guo, Guangwen Li, Xiaoqing Ma, Haiping Zhao, Junfen Fan, Tianwei Liu, Yumin Luo, Yunliang Guo

Abstract

Aims: Inflammatory processes induced by leukocytes are crucially involved in the pathophysiology of acute ischemic stroke. This study aimed to elucidate the inflammatory mechanism of long non-coding RNA (lncRNA) H19-mediated regulation of C1q and tumor necrosis factor 6 (C1QTNF6) by sponging miR-29b in leukocytes during ischemic stroke.

Methods: H19 and miR-29b expression in leukocytes of patients with ischemic stroke and rats with middle cerebral artery occlusion were measured by real-time polymerase chain reaction. H19 siRNA and miR-29b antagomir were used to knock down H19 and miR-29b, respectively. We performed in vivo and in vitro experiments to determine the impact of H19 and miR-29b on C1QTNF6 expression in leukocytes after ischemic injury.

Results: H19 and C1QTNF6 upregulation, as well as miR-29b downregulation, was detected in leukocytes of patients with stroke. Moreover, miR-29b could bind C1QTNF6 mRNA and repress its expression, while H19 could sponge miR-29b to maintain C1QTNF6 expression. C1QTNF6 overexpression promoted the release of IL-1β and TNF-α in leukocytes, further exacerbated blood-brain barrier disruption, and aggravated the cerebral ischemic injury.

Conclusions: Our findings confirm that H19 promotes leukocyte inflammation by targeting the miR-29b/C1QTNF6 axis in cerebral ischemic injury.

Trial registration: ClinicalTrials.gov NCT03577093.

Keywords: C1QTNF6; inflammation; ischemic stroke; leukocyte.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2022 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.

Figures

FIGURE 1
FIGURE 1
H19, miR‐29b, and C1QTNF6 expression in patients with acute stroke. (A) Quantitative RT‐PCR results for H19, miR‐29b, and C1QTNF6 levels in neutrophils of patients with acute stroke. Acute ischemic stroke group (N = 50) and control group (N = 42). (B) Correlation between miR‐29b and C1QTNF6 expression levels at admission (N = 50). (C) Correlation of H19 and miR‐29b expression with NIHSS score at admission (N = 50). (D) The predicted binding sites between miR‐29b and C1QTNF6 and between miR‐29b and H19. (E) Plasma TNF‐α, IL‐1β, and MMP‐9 levels in patients with acute ischemic stroke (N = 50). *p < 0.05, ***p < 0.001 vs. control group
FIGURE 2
FIGURE 2
Changes in H19 and miR‐29b levels and subsequent effects on cerebral injury and neurological deficits in MCAO rats. (A) H19 and miR‐29b expression levels in leukocytes and ischemic brain tissues after MCAO injury (N = 9). (B) Quantitative RT‐PCR experiment to confirm the efficacy of H19 siRNA and miR‐29b antagomir transfection (N = 9). (C) Effects of intravenous injections of H19 siRNA and miR‐29b antagomir on cerebral injury and neurological function deficits at 24 h after stroke (N = 12). (D and E) Neuronal apoptosis in the ipsilateral cortex as detected by NeuN/TUNEL immunofluorescence double staining (N = 6). *p < 0.01, ***p < 0.001 vs. sham group. #p < 0.05, ###p < 0.001 vs. MCAO group. Scale bar = 20 μm
FIGURE 3
FIGURE 3
H19 and miR‐29b regulate the expression of C1QTNF6 in the leukocytes of MCAO rats. (A) Leukocyte C1QTNF6, IL‐1β, and TNF‐α protein levels assessed by Western blotting (N = 6). (B) Ipsilateral brain tissue C1QTNF6, IL‐1β, and TNF‐α protein levels assessed by Western blotting (N = 6). *p < 0.05 vs. sham group; #p < 0.05, vs. MCAO group
FIGURE 4
FIGURE 4
H19 siRNA reverses the miR‐29b antagomir‐aggravated acute cerebral ischemic injury. (A) Cerebral infarct volume and brain edema evaluated by TTC staining of coronal brain sections (N = 12). (B) Neuronal apoptosis in the ipsilateral cortex as detected by NeuN/TUNEL immunofluorescence double staining (N = 6). (C) Leukocyte C1QTNF6 mRNA, IL‐1β, and TNF‐α protein levels assessed by quantitative RT‐PCR and Western blotting (N = 6). (D) Ipsilateral brain tissue C1QTNF6, IL‐1β, and TNF‐α protein levels assessed by Western blotting (N = 6). &p < 0.05, &&&p < 0.001 vs. MCAO+miR‐29b antagomir+H19 siRNA control group. Scale bar = 20 μm
FIGURE 5
FIGURE 5
H19 and miR‐29b regulate ZO‐1 and occludin expression in hCMEC/D3 cells. (A and B) hCMEC/D3 cell apoptosis induced by H19 siRNA and miR‐29b antagomir treatment (N = 6). (C) C1QTNF6, IL‐1β, and TNF‐α protein levels in HL‐60 cells assessed by Western blotting (N = 6). (D) ZO‐1 and occludin protein levels in hCMEC/D3 cells assessed by Western blotting (N = 6). *p < 0.05 vs. sham group; ***p < 0.001 vs. sham group; #p < 0.05 vs. OGD+vehicle‐siRNA group or vehicle‐siRNA group; ##p < 0.01 vs. OGD+vehicle‐siRNA group or vehicle‐siRNA group; &p < 0.05 vs. OGD+miR‐29b antagomir group; &&&p < 0.001 vs. OGD+miR‐29b antagomir group

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