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

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.

Peripheral leukocytes significantly affect the clinical outcome of stroke and can be a therapeutic target to treat and prevent ischemic stroke. [4][5][6] There have been several studies on the expression of non-coding ribonucleic acids (ncRNAs) in peripheral leukocytes of patients with ischemic stroke. The ncRNAs in leukocytes regulate the release of inflammatory factors to promote inflammatory responses after cerebral ischemic injury. 7 They can be classified as short (≤200 base pairs) or long ncRNAs (lncRNAs; >200 base pairs). 8 MicroRNAs (miRNAs or miRs) are ncRNAs with <22 nucleotides that modulate protein expression at the post-transcriptional level. 4

miR-
NAs prevent messenger RNA (mRNA) translation by binding to the consensus seed sequences in the 3′ untranslated regions (UTRs) of target mRNAs. 4 Recent studies have demonstrated mutual regulation between miRNAs and lncRNAs, including a direct interaction and an indirect role by binding to target genes in ischemic stroke. 9 To identify the leukocyte genes related to ischemic stroke, we previously analyzed mRNA expression profiles of neutrophils using RNA-seq and compared the profiles of patients with acute ischemic stroke and healthy controls. We found an alteration in the expression of C1q and tumor necrosis factor 6 (C1QTNF6) after ischemic stroke. C1QTNF6 was originally identified in the heart, brain, and peripheral inflammatory cells. It is known to be involved in diverse processes, including apoptosis, metabolism, and release of inflammatory factors. 10 C1QTNF6 can directly modulate pro-inflammatory cytokine gene expression in leukocytes. 11 Our preliminary findings demonstrated that C1QTNF6 mRNA could be modulated by several miRNAs in neutrophils, including miR-29b. The expression of miR-29b in leukocytes is significantly decreased after ischemic injury, and changes in the miR-29b level are correlated with the prognosis of ischemic stroke. 12 In contrast, miR-29b overexpression alleviates brain injury and attenuates blood-brain barrier (BBB) damage. 12 We predicted the potential targeting lncRNAs related to miR-29b and identified the binding site of miR-29b in H19 using StarBase.
According to a report, H19 levels increase in the circulating blood after ischemic stroke, which promotes the release of inflammatory factors. 13 Therefore, we hypothesized that C1QTNF6 in leukocytes might be regulated by H19 and miR-29b that are probably involved in the pathogenesis of ischemic stroke.
This study aimed to elucidate the inflammatory mechanism of lncRNA H19-mediated regulation of C1QTNF6 by sponging miR-29b in leukocytes during ischemic stroke.
We recruited 50 patients with acute ischemic stroke of anterior circulation from the emergency department of Xuanwu Hospital between March 2016 and December 2017. Ischemic stroke was diagnosed by two neurologists based on the patient history, laboratory examinations, neurological deficits, and diffusion-weighted magnetic resonance imaging (MRI) findings. We included patients who had ischemic stroke for the first time, aged 18-80 years, had a National Institute of Health Stroke Scale (NIHSS) score <25 points and were admitted within 6 h after the onset of symptoms. For the control group, we included 42 age-and sex-matched healthy volunteers from the medical examination center who did not have any central nervous system disease.

| Blood collection and separation of neutrophils
Venous blood samples were collected from patients with ischemic stroke and healthy controls in vacuum tubes containing ethylene diamine tetra-acetic acid and anticoagulant upon admission. Blood samples were fractionated by centrifugation at 3000 g for 10 min at 4°C. Subsequently, the plasma layer was aliquoted and stored at −80°C for routine laboratory assays. Neutrophils were separated using a standard Ficoll-Paque Plus gradient method for RNA extraction and examined as described in a previous report. 4

| Animals
All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Qingdao University. Animal studies are conducted in compliance with the ARRIVE guidelines 2.0. 14 Male Sprague-Dawley rats weighing 260-280 g were purchased from Vital River Laboratory Animal Technology Co. Ltd. The animals were maintained in standard-housing and open-top cages in a pathogen-free facility at 23 ± 1°C with 50%-60% humidity at Qingdao University. All animals underwent fasting for 24 h with free access to tap water before the experimental procedures.

| Middle cerebral artery occlusion
Rats underwent surgery for right middle cerebral artery occlusion (MCAO). After anesthetization with enflurane, a nylon filament with a silicon tip of 0.37 mm diameter was inserted into the right middle cerebral artery to obstruct blood flow for 2 h. Subsequently, it was removed to allow reperfusion. Regional cerebral blood flow was observed using transcranial laser Doppler to confirm a decrease in blood flow by 20%-30%, compared with the baseline value during MCAO. Sham-operated rats underwent the same anesthetic and surgical procedures without MCAO.

| Behavioral tests
Behavioral tests were performed using 12 animals in each group.
Rats underwent neurological evaluation using a Zea-Longa 5-point scoring system before and 24 h after surgery. 15

| 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining
Rats were sacrificed 24 h after reperfusion. The brains were quickly removed and cut into 2 mm thick coronal slices for TTC staining. The infarct volume was calculated as follows: Volume calculation with edema correction was as follows:
Proteins were detected by incubation with horseradish peroxidaseconjugated secondary antibodies (Santa Cruz Biotechnology) for 60 min at room temperature, using an enhanced chemiluminescence kit (Millipore). Gray values of the protein bands were analyzed using AlphaEase FC software (Alpha Innotech).

| Real-time polymerase chain reaction (RT-PCR)
Equal quantity of purified RNA was obtained from neutrophils, leukocytes, and brain tissues and used as a template for cDNA synthesis using SYBR Green qPCR Master Mix

| NeuN/TUNEL staining and immunofluorescence analysis
Rats were killed through chloral hydrate administration, and cardiac perfusion was performed with physiological saline and 4% paraformaldehyde. The brain was harvested and fixed in 4% paraformaldehyde for 48 h, followed by dehydration in 30% sucrose. Fluorescence images were acquired using an Olympus Fluoview FV1000 fluorescence microscope (Olympus).

| ELISA
Plasma samples were collected from stroke patients and healthy volunteers as previously described. 4 Approximately 200 μl of plasma was prepared in an ice-cold PBS. TNFα, IL-1β, and MMP-9 protein levels were quantified using a commercial ELISA kit (Abcam), following the instructions of the manufacturer.
containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in an atmosphere of 5% CO 2 . HL-60 cells were transfected with a mixture of H19 siRNA, miR-29b antagomir or control, and Lipofectamine RNAiMAX (GenePharma). The cells were incubated for 24 h in a humidified incubator for further analyses.
Human brain capillary endothelial cells (hCMEC/D3) were grown in EBM-2 medium supplemented with vascular endothelial growth factor, insulin-like growth factor-1, epidermal growth factor, basic fibroblast growth factor, hydrocortisone, ascorbate, penicillin-streptomycin, and 2.5% fetal calf serum. The cells were maintained in an incubator at 37°C and 5% CO 2 . Oxygen-glucose deprivation (OGD) and reoxygenation were used as in vitro models for mimicking cerebral ischemia. Cultured hCMEC/D3 cells were kept in a glucose-free and hypoxic incubator chamber with a gas mixture of 94.5% N 2 , 0.5% O 2 , and 5% CO 2 at 37°C for 2.

| Flow cytometry
The apoptosis ratio was estimated using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 for flow cytometry, following the instructions of the manufacturer (Invitrogen). Cells were collected, washed twice with cold PBS, and incubated with Annexin V-FITC mixed with propidium iodide for 10 min in the dark. Cellular fluorescence was assessed using flow cytometry (CytoFLEX S, Beckman).

| Statistical analysis
Statistical analyses were performed using SPSS version 23.0.
Shapiro-Wilk test to test the normality of the data. Normally distributed data are expressed as mean ± SD. Student's t-test was used for between-group comparisons. One-way analysis of variance with the Tukey-Kramer post hoc test was used to compare several quantitative variables. Non-normally distributed data were evaluated using the Mann-Whitney U-test and were expressed as medians.
Pearson's correlation test was used to assess between-variable correlations. Statistical significance was set at p < 0.05.

| Upregulation of H19 and C1QTNF6 and downregulation of miR-29b in neutrophils of patients with ischemic stroke
We investigated a novel gene, C1QTNF6, which demonstrated increased mRNA levels in neutrophils of patients with acute ischemic stroke compared with healthy volunteers ( Figure 1A, p < 0.05).
Further analysis using TargetScan and StarBase databases revealed that C1QTNF6 is regulated by miR-29b and H19. We identified the putative miR-29b binding sites of H19 and 3′ UTR of C1QTNF6 mRNA ( Figure 1D). To confirm this, we conducted quantitative RT-PCR to evaluate mRNA expression levels of miR-29b, H19, and C1QTNF6 in neutrophils. H19 expression in neutrophils was significantly increased in patients with stroke ( Figure 1A, p < 0.05). Additionally, the patients had decreased miR-29b expression ( Figure 1A, p < 0.05), which significantly increased the C1QTNF6 levels. There was a negative linear correlation between miR-29b and C1QTNF6 levels in the neutrophils of patients with stroke ( Figure 1B, r = −0.716, p < 0.001).

| H19 and miR-29b levels correlate with the NIHSS score
To MMP-9 levels were significantly higher in the patients than in the controls ( Figure 1E, p < 0.05). These results suggest that H19 and miR-29b in neutrophils are critically involved in ischemic stroke.

| H19 expression is upregulated after MCAO injury, while H19 inhibition alleviates MCAO injury in rats
Considering the increased expression of H19 in patients with stroke, we further evaluated the role of H19 in cerebral ischemic injury in rats by intravenous injection of H19 siRNA three days before MCAO surgery. Quantitative RT-PCR was performed to detect H19 expression and to verify the efficacy of H19 siRNA transfection.
Initially, the level of H19 in the ischemic brain of rats was increased

| MiR-29b expression is downregulated after MCAO injury, and miR-29b inhibition exacerbates cerebral injury
We investigated the effect of intravenous injection of miR-29b antagomir by measuring the infarct volume and neurological deficits after MCAO injury. First, we found decreased levels of miR-29b in the ischemic brain and leukocytes in MCAO rats at 24 h after the injury (Figure 2A
We further analyzed IL-1β and TNFα expression in MCAO rats

| H19 inhibition reverses MCAO injury, which is worsened by miR-29b antagomir
To further elucidate the relationship among H19, miR-29b, and C1QTNF6 in cerebral ischemic injury, we intravenously injected H19 siRNA in MCAO rats with miR-29b antagomir and analyzed the infarct volume and brain edema at 24 h after MCAO injury.
H19 siRNA reduced the cerebral infarct volume and brain edema compared with that in MCAO rats treated with miR-29b antagomir

| H19 and miR-29b in HL-60 cells regulate hCMEC/D3 apoptosis
To explore whether leukocyte C1QTNF6 affects hypoxia-induced BBB disruption through an inflammatory response, we established an in vitro model in which OGD-exposed hCMEC/D3 cells were co-cultured with HL-60 cells. HL-60 cells in different groups were ( Figure 5D). These findings suggest that H19 in leukocytes aggravates hypoxia-induced hCMEC/D3 apoptosis by targeting the miR-29b/C1QTNF6 axis.

| DISCUSS ION
Our findings confirmed the upregulation of H19 in neutrophils of patients with acute ischemic stroke, which is positively associated with the severity of neurological deficits. Moreover, we observed miR-29b downregulation in the neutrophils of ischemic stroke patients, which was negatively associated with H19 levels. Additionally, we demonstrated that H19 regulates C1QTNF6 expression by sponging miR-29b, which facilitates the release of IL-1β and TNFα by leukocytes and BBB disruption during cerebral ischemic injury. Our findings provide new insights for possible targeted treatments of ischemic stroke.
Emerging evidence regarding peripheral immune cells has provided insights into novel inflammatory mechanisms contributing to neuronal cell death in cerebral ischemic injury. 16,17 Leukocytes are sensitive indicators of inflammatory stimuli in patients with ischemic stroke. 18,19 There is altered leukocyte expression of multiple ncRNAs in patients with ischemic stroke, including H19, which is crucially involved in the inflammatory response of ischemic stroke. 13 We previously reported that H19 increases the immune response by increasing plasma TNFα and IL-1β levels after ischemic stroke; moreover, it affects the subsequent pathological outcome. 13  Multiple studies have demonstrated that miR-29b is a key mediator of ischemic cascade in regulating inflammation and neuronal apoptosis. 23 There is a decreased miR-29b expression in ischemic regions and OGD-activated microglia, which contributes to injury in focally ischemic brains. 24 Additionally, miR-29b can inhibit neuronal apoptosis by suppressing TNFα and IL-1β release in OGD-activated microglia. 24 The miR-29b level in the blood of patients significantly F I G U R E 3 H19 and miR-29b regulate the expression of C1QTNF6 in the leukocytes of MCAO rats. (A) Leukocyte C1QTNF6, IL-1β, and TNFα protein levels assessed by Western blotting (N = 6). (B) Ipsilateral brain tissue C1QTNF6, IL-1β, and TNFα protein levels assessed by Western blotting (N = 6). *p < 0.05 vs. sham group; # p < 0.05, vs. MCAO group decreased with ischemic stroke and in the blood and brain of patients with cerebral ischemic injury. 12 This suggests that miR-29b in the brain and circulating blood could be involved in regulating the post-stroke immune response, which is consistent with our findings.
We found that miR-29b excision promoted neuronal apoptosis and upregulated TNFα and IL-1β expression by targeting C1QTNF6. and TNFα, which promotes neutrophil recruitment to the brain parenchyma and exacerbates inflammation. 31 We observed increased IL-1β and TNFα secretion after C1QTNF6 overexpression in the peripheral leukocytes of MCAO rats. Additionally, increased IL-1β and TNFα levels in the brain promote leukocyte migration into the brain parenchyma and increase BBB permeability, which is a vicious cycle. Consistent with our in vivo study, C1QTNF6 overexpression

| CON CLUS IONS
In summary, this is the first study to demonstrate H19 and C1QTNF6 upregulation and miR-29b downregulation in leukocytes of patients with ischemic stroke and MCAO rats. H19 promotes C1QTNF6 expression by directly targeting miR-29b in leukocytes of MCAO rats.
Furthermore, C1QTNF6 upregulation in leukocytes aggravates cerebral ischemic injury and promotes BBB damage through IL-1β and TNFα upregulation. Our findings demonstrate the inflammationmodulatory effect of the H19/miR-29b/C1QTNF6 axis in the mechanism of cerebral ischemia injury.

ACK N OWLED G M ENTS
We would like to thank Editage (www.edita ge.cn) for English language editing.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.