Long non‐coding RNA MALAT1 regulates angiogenesis following oxygen‐glucose deprivation/reoxygenation

Abstract Long non‐coding RNAs (lncRNAs) have been identified as playing critical roles in multiple diseases. However, little is known regarding their roles and mechanisms in post‐stroke angiogenesis. Our studies focused on deciphering the functional roles and the underlying mechanisms of the lncRNA metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) in the process of angiogenesis following oxygen‐glucose deprivation/reoxygenation (OGD/R). We characterized the up‐regulation of MALAT1 expression in the process of angiogenesis after hypoxic injury in vivo and in vitro. We further showed that compared with the empty vector, MALAT1 knockdown had significantly reduced the capacity for angiogenesis, which was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium (MTT), scratching, cell cycle and immunofluorescent staining. Thus, our findings suggest that MALAT1 may mediate proangiogenic function in OGD/R. To further explore the potential mechanisms, we used lentiviruses expressing shMALAT1 and empty vector; the results revealed that shMALAT1 reduced the expression of 15‐lipoxygenase 1 (15‐LOX1), vascular endothelial growth factor (VEGF) and the phosphorylation of signal transducers and activators of transcription 3 (pSTAT3). Taken together, our results are the first to propose that MALAT1 may regulate angiogenesis through the 15‐LOX1/STAT3 signalling pathway, and they may provide a critical target for the treatment of hypoxic injury and an avenue for therapeutic angiogenesis.


| INTRODUC TI ON
Stroke has been shown to be one of the leading causes of mortality and the most common cause of disability in adults in most countries. 1 Although clinically effective drug (recombinant tissue plasminogen activator, rt-PA) therapy has been achieved for acute ischaemic stroke, the majority of patients miss the optimal opportunity for vascular recanalization, owing to the narrow therapeutic window and safety concerns. 2 Thus, there remains an urgent need to decipher the potential mechanisms of stroke, understanding those mechanisms will be beneficial for the clinical therapy of patients.
A novel class of non-coding RNAs over 200 nucleotides in length, known as long non-coding RNAs (lncRNAs), is pervasively transcribed in the mammalian genome. 3 Within the past decade, aberrantly expressed lncRNAs have been demonstrated to act as key regulators of post-transcriptional gene expression in pathological aspects of ischaemic stroke. 4 However, less is known about the participation of lncRNAs in regulatory mechanisms of angiogenesis following cerebral I/R. MALAT1 is a highly abundant and evolutionary conserved lncRNA that was first described as being associated with the metastasis of lung tumours. 5 Recent studies have shown that MALAT1 is closely associated with endothelial cell repair after ischaemia. 6,7 Furthermore, in a remarkable discovery by Zhang et al., RNA-sequencing revealed that MALAT1 is significantly up-regulated in oxygen-glucose deprivation (OGD)-responsive endothelial cells. 8 However, the biological function and molecular mechanisms of action of MALAT1 in angiogenesis following I/R have not been previously reported. Therefore, we will evaluate whether MALAT1 is involved in angiogenesis after cerebral I/R and if yes, what is the detail mechanism.

| Focal cerebral ischaemia
Mice were anaesthetized with 1.5% isoflurane in 30% oxygen with a face mask. After a midline to the right skin incision, the right common carotid artery and the internal carotid artery were exposed and the branches of the common carotid artery were electrocoagulated. (F) Statistical analysis of the mean density of VEGF (n = 7/group). Data are presented as the mean ± SEM. ***P < 0.001 vs sham group length of 7-0 rounded silicone-rubber-coated monofilament (L3600, Jialing, Guangzhou, China) was introduced from the common carotid artery up to internal carotid artery until regional cerebral blood flow (rCBF) reduction (>70%). 9 After 60 minutes, the filament was removed to allow reperfusion for 24 hours. Sham group animals were subjected to similar operations to expose the internal carotid artery but without the occlusion of the middle cerebral artery. Subsequently, the Zea Longa score was used to determine the degree of neurological deficit. 10 Zea Longa scores are divided into 5-grade, as shown in Table 1.

| Measurement of cerebral infarction volume
Mice were given an overdose of anaesthesia with isoflurane and decapitated (n = 8/group) 24 hours after reperfusion. The brains were carefully removed, and 2-mm-thick coronal sections were collected in 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, USA) for 15 minutes at 37°C, followed by overnight fixation in 4% paraformaldehyde (pH 7.4) (Solarbio, Beijing, China). 11 Sections were imaged and digitized. Then, the images were analysed with ImageJ software to determine the infarct area. Infarct size was expressed as a percentage of hemispheric area. 12

| Immunohistochemical analysis
Histological sections (4 μm in thickness) prepared from paraffin-embedded tissue samples of ischaemic brains were used for the immunohistochemical analysis. Sections were blocked using 0.3% H 2 O 2 for 10 minutes.
Subsequently, the sections were incubated with anti-VEGF primary antibody (1:100, ab1316, Abcam, Cambridge, MA, USA) for 2 hours at room temperature. The brain tissues were then rinsed with phosphate-buffered saline (PBS). Next, the sections were incubated with biotinylated goat anti-mouse IgG secondary antibody (Zsbio, Beijing, China) for 30 minutes.
3,3-Diaminobenzidine (DAB) was used as the chromogen. No primary antibody was used in the secondary only control. After staining, the mean density of VEGF was evaluated in three randomly selected fields per section under a microscope (Nikon, Tokyo, Japan).

| Fluorescence in situ hybridization (FISH)
The techniques and labelled single-stranded RNA probes used for in situ hybridization were used as previously described. 13 The expression of MALAT1 was determined according to the instructions of the fluorescence in situ hybridization (FISH) kit used in our studies (Boster, Wuhan, China). Hybridization was performed on cryostat-cut coronal brain sections (10 μm) by incubating with a labelled RNA probe overnight at 48°C. The sections were washed, dehydrated, coated, developed and exposure.

| Cell cultures
A mouse brain microvascular endothelial cell line was purchased from Fuheng (Shanghai, China) and it was certified by Biosystems.
The cells were incubated in six-well plates (Corning, New York, USA

| OGD/R model of ECs
Ischaemia was mimicked in vitro as follows: first, cells were washed with PBS (Solarbio, Beijing, China). Next, the cells were cultured in glucose-free DMEM (GIBCO, New York, USA) without FBS in a hypoxia incubator (95% N 2 , 5% CO 2 ) at 37°C for 10 hours. After OGD exposure, the medium was replaced with absolute medium containing 10% FBS and cultured under normoxic conditions (95% air, 5% CO 2 , 21% FiO 2 ) to perform OGD/R model. Cells were incubated at 37°C in normal-glucose DMEM under normoxic conditions as a control.
Importantly, the hypoxic chamber was previously sealed and placed in the 37°C thermostat container (SPX-150C, Boxun, Shanghai, China), where it was flushed in advance with a gas mixture of 95% N 2 , 5% CO 2 for 30 min at the rate of 2 L/min. After flushing, the concentration of O 2 was managed with a gas monitor (Smart Sensor, Hong Kong, China).
The concentration of O 2 was maintained at less than 1%.

| Cell viability assay
MTT (Beyotime, Beijing, China) was used to evaluate cellular viability according to the manufacturer's instructions. Cells at a seeding rate of per 2 × 10 4 well were incubated in 96-well plates and added to culture medium with 10 μL of 5 mg/mL MTT reagent at 37°C for 4 hours under normal growth conditions. Then, the cells were lysed at room temperature by adding 100 μL of dimethyl sulfoxide (DMSO, Solarbio, Beijing, China) for 10 minutes. Finally, cell viability was determined by measuring the optical density (OD) at 490 nm in a universal enzyme marker (Bio-Rad, Shimadzu, Japan).
Percent change relative to the control was calculated as a measure of cell viability.

| Immunofluorescent staining analysis
Cells were cultured on coverslips in 24-well plates and then fixed with 4% paraformaldehyde for 20 minutes at room temperature.

| Cell migration
Migration was assessed using a scratching assay. A six-well plate was divided with a marker pen approximately every 0.5-1.0 cm. A total of 5 × 10 5 cells per well were incubated in a six-well plate and grown to confluence overnight. In this confluent monolayer, a single scarification was made with 200-μL plastic pipette tip. The healing of the scratch was visualized at the indicated time (48 hours) using a microscope (×4). The area of the scratch was analysed using ImageJ software.

| Quantitative real-time polymerase chain reaction
Total RNA was extracted from I/R cerebral tissue or OGD/R cells  Table 2.

| Cell transfection
To generate a lentivirus expressing shMALAT1 and an empty vector, we cultured the cells in 6-well plates for 12 hours, the culture medium was centrifuged to collect the lentivirus. A volume of 300 μL of each virus supernatant was added into 700 μL of fresh culture with 1 μL of polybrene (final concentration degree 8 ng/mL). The premixed viral infection was added to a new culture dish containing cells at no more 60% confluence. The culture medium was changed the following day. After 48 hours, the cells were screened with puromycin (Thermo Fisher, Shanghai, China) and then cultured for another 2 days. When the cells were harvested, some were frozen and the rest were used for the subsequent experiments.

| Enzyme-linked immunosorbent assay (ELISA)
The concentration of 15-HETE was evaluated using an ELISA according to the manufacturer's instruction, as described previously. 16 In order to measure the concentration of 15-HETE after inhibition, the 15-HETE kit (SinoBest, Shanghai, China) was employed. The calibration standards are assayed at the same time as the sample and the operator was allowed to produce a standard curve of OD vs 15-HETE concentration.

The concentration of 15-HETE in the samples is then determined by
comparing the OD of the samples to the standard curve.

| Western blot analysis
Samples from cerebral tissues and cells were homogenized in lysis buffers (Solarbio, Beijing, China) with a phosphorylation inhibitor (Roche, Indianapolis, USA), and total protein was isolated as described previously. 17 The total protein concentration for each sam- The survival rate, proliferation and migration of brain microvascular endothelial cells between the oxygen-glucose deprivation/reoxygenation (OGD/R) and control groups. Finally, the antigen-antibody complexes were detected using an enhanced chemiluminescence (ECL) reagent kit (Vazyme, Nanjing, China). The intensity of each band area was quantified using ImageJ.

| Statistical analysis
Data are expressed as the mean ± standard error (SE), except for the neurological deficit scores, which are presented as the medi-

| Angiogenesis is activated after ischaemic injury
Sixty C57BL/6J mice were subjected to middle cerebral artery occlusion MCAO 1 hour before reperfusion for 24 hours; six mice died and the other fifty-four mice survived. We evaluated the ischaemic infarct volumes, and mice were randomly selected for TTC staining after surgery. Infarct volume was significantly increased in the MCAO group compared with the sham group ( Figure 1B).
The neurological deficit scores were estimated in the sham and MCAO groups ( Figure 1C). Furthermore, we employed VEGF immunohistochemistry to analyse the amount of microvasculature in the ischaemic penumbra 24 hours following cerebral ischaemic stroke. The results demonstrated that VEGF was up-regulated in the MCAO group compared with sham group (Figure 1D), indicating that angiogenesis is activated upon ischaemic injury.

| OGD/R promotes ECs proliferation, migration and CD31 positive cell expression
Brain microvascular endothelial cells were cultured to confluence, and then subjected to OGD for 10 hours. Next, they were  (Figure 2A). Consequently, we selected reoxygenation for 48 h for subsequent assays. Accordingly, the functional analysis associated with angiogenesis of ECs following OGD/R, including the cell proliferation assay ( Figure 2B,C), migration assay ( Figure 2D,E) and CD31 + microvessel counts by immunofluorescence ( Figure 2F,G), also showed significant elevations in OGD/R-treated cells compared with the control group.
Therefore, the proliferation, migration and proangiogenic capacity of ECs exposed to OGD/R increased.

| MALAT1 is an angiogenesis-associated lncRNA
To investigate the role of MALAT1 in the processes of angiogenesis following I/R, total RNA was extracted from the ischaemic penumbra following I/R. Then, we examined MALAT1 expression. We found that MALAT1 expression was aberrantly up-regulated in I/R ( Figure 3A). Moreover, we observed that the expression of MALAT1 in ECs was significantly increased in response to OGD/R in vitro ( Figure 3B). Taken together, these results show that ischaemia or hypoxia can induce the up-regulation of MALAT1 both in vivo and in vitro.
In addition, MALAT1 expression was mostly located in the nuclei of cells according to FISH. The MALAT1 expression level was significantly greater in the MCAO group than that in the sham group ( Figure 3C). Moreover, we found that the expression of angiogenesis-associated marker CD31, which was located in the endothelial cells of microvessels, and showed a trend similar to that of MALAT1 in the sham and MCAO groups ( Figure 3C,D). The results of FISH confirmed that MALAT1 expression may affect CD31 expression in murine cerebral tissues. Therefore, the experimental evidence supported our hypothesis and demonstrated that MALAT1 may be involved in ischaemia or hypoxia/reperfusion induced angiogenesis of cerebrovascular endothelial cells.

| Knockdown of MALAT1 reduces ECs proliferation and migration
To assess the potential function of MALAT1 in the biological processes of angiogenesis, we studied the functional significance of MALAT1 alteration in vitro. Brain microvascular endothelial cells were transfected with a lentivirus to cause a marked reduction in the level of MALAT1 ( Figure 3E). Although the reduction of MALAT1 expression was significant in both interference fragments, it was more effectively reduced in the second interference fragment ( Figure 3E). Accordingly, we used the second fragment in the subsequent assays. We conducted MTT and cell cycle assays, and found that the cell viability ( Figure 4A) and proliferation ( Figure 4B,C) were markedly repressed in ECs with MALAT1 knockdown compared with empty vector group. Similarly, the scratching assay showed that decreased MALAT1 impaired the migration capacity of ECs ( Figure 4D,E). The results demonstrate that MALAT1 can mediate ECs proliferation and contribute to cell migration.

| MALAT1 promotes CD 31-positive endothelial cell expression
To further investigate the effect of MALAT1 on endothelial cells following OGD/R, we employed immunofluorescent staining and found that the knockdown of MALAT1 markedly reduced the number of CD31-positive ECs compared to the number in the blank control group ( Figure 4F,G). These results suggest that MALAT1 can promote the formation of microvessels in response to OGD/R.

| D ISCUSS I ON
Stroke, as a cerebrovascular accident, causes a loss of brain function due to a disturbance in the blood supply to the brain. Following stroke, the affected area of the brain cannot function normally, which may result in disability and even death. 21 Substantial efforts are still being devoted to deciphering the complex mechanisms of stroke. Interestingly, accumulated studies have shown that angiogenesis is activated after stroke and that higher neovascular density is associated with less morbidity, disability and mortality. 5 Therefore, angiogenesis has been recognized as a key to the recovery of brain function. 5 LncRNAs have been demonstrated to be one of the most abundant classes of ncRNAs. 22 As the versatile roles of lncRNAs in biological processes and human disorders are increasingly recognized, these RNAs are attracting more extensive attention in the fields of molecular biology and clinic research. 23 Furthermore, lncRNAs are reported to be potential diagnostic biomarkers and therapeutic targets for multiple diseases . 24 In particular, lncRNAs play a role as a novel type of master regulator after ischaemic stroke. MALAT1 was initially recognized as a tumour-associated lncRNA-mediating cancer metastasis and cell survival. 25,26 Although there is no direct evidence Data are presented as the mean ± SEM. *P < 0.05 vs control, ***P < 0.001 vs control,***P <0.001 vs control STAT3 has also been demonstrated to be an important player in cellular processes including proliferation and migration. 19,25 Many studies have reported that STAT3 is activated and translocated into the nucleus upon exposure to hypoxia. Its increased expression and activation may be linked to cardiovascular diseases, such as atherosclerosis and stroke. STAT3 has also been demonstrated to mediate VSMC growth and migration in response to ischaemic injury 37,38 Moreover, STAT3-mediated cerebral ischaemic tolerance has been well characterized and can affect the brain parenchyma and vasculature associated cells, providing not only neuroprotection but also cerebral blood flow in the presence of pathology. The knockdown of STAT3 increases the injury to the brain. 39   The concentration of 15-HETE (pg/mL) was determined by enzyme-linked immunosorbent assay. Data are presented as the mean ± SEM.*P < 0.05 vs oxygenglucose deprivation/reoxygenation (OGD/R), **P < 0.01 vs OGD/R, ***P < 0.001 vs OGD/R potential targets of MALAT1 and the functional roles of some miRNAs between MALAT1 and 15-LOX1 or STAT3 were not investigated in this article and still need to be further studied.

| CON CLUS IONS
In summary, our findings are the first to demonstrate that the angiogenesis-associated lncRNA MALAT1 exerts proangiogenic effects through the 15-LOX1/STAT3 signalling pathway in I/R and OGD/R, which provides a detailed understanding of angiogenesis after OGD/R injury. Therefore, MALAT1 expression is augmented in brain endothelial cells under ischaemia, which may contribute to a potential therapeutic strategy for patients who have suffered ischaemic stroke.

ACK N OWLED G EM ENT
This research has not received specific grants from any public funding agency.

D I SCLOS U R E
There is no conflict of interests.