Knockdown of MALAT1 attenuates pyroptosis in diabetic cerebral ischemia via STAT1

Nan Zhao First Clinical Hospital, Harbin Medical University Wei Hua First Clinical Hospital, Harbin Medical University Qi Liu First Clinical Hospital, Harbin Medical University Yueying Wang First Clinical Hospital, Harbin Medical University Zhiyi Liu First Clinical Hospital, Harbin Medical University Sinan Jin First Clinical Hospital, Harbin Medical University Benshuai Wang First Clinical Hospital, Harbin Medical University Yuxin Pang First A liated Hospital of Harbin Medical University Jiping Qi First Clinical Hospital, Harbin Medical University Yuejia Song (  drsongyuejia@hotmail.com ) No. 23 Post Street, Nangang District https://orcid.org/0000-0002-8919-7801


Introduction
Cerebral ischemia is featured by high mortality and disability rates. Over 30% of ischemic patients are diagnosed with diabetic disease [1]. Diabetes is reported to be an independent risk factor for stroke; meanwhile, the morbidity of cerebral ischemia patients with diabetic disease is 2 to 4 times to the patients without [2][3][4]. In ammation is closely related to the occurrence and development of cerebral ischemia, mainly involving brain edema, the destruction of the blood-brain barrier, the in ltration of in ammatory cells, and the expression of adhesion molecules [6]. However, the speci c mechanism is still unclear. The current therapies for cerebral ischemia are chie y based on increasing perfusion, but it might develop into ischemia/reperfusion (I/R) injury [7]. Therefore, to further explore therapeutic approaches to ameliorate cerebral ischemic injury is a pivotal issue.
Pyroptosis, also known as in ammatory cell death, is a programmed cell death rst identi ed by Brennan and Cooksen in 2000 [8]. It is triggered by a series of pattern recognition receptors (PRRs): activated NLRP3 in ammasomes, amino-terminal gasdermin D (GSDMD-N) with pore-forming activity. Next, the activated GSDMD-N binds to the plasma membrane and forms large oligomeric pores, releasing cell contents and pro-in ammatory factors (IL-1β, IL-18). In ammatory response is an essential pathogenesis of diabetes. Furthermore, pyroptosis expands the in ammatory effects and participates in the progression of numerous diabetic complications, including periodontitis [9], nephropathy [10], retinopathy [11] and cardiomyopathy [12]. Tu et al. [13] revealed that the neuronal pyroptosis in diabetic patients is remarkably increased compared with the non-diabetic ischemic stroke. Therefore, the pyroptosis of neurons makes limited contributions to the establishment of the diversity, stability and plasticity in brain nervous system.
Long noncoding RNAs (lncRNAs) are involved in various physiology and pathology progressions, including pyroptosis. With the deepening research of lncRNAs, lncRNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) has been revealed to aggravate ischemic stroke via the MDM2/p53 [14][15][16]. Besides, MALAT1 performed regulatory functions on pyroptosis in diabetic complications. For instance, knockdown of MALAT1 exhibited suppressive effects on macrophage pyroptosis in rats with diabetic atherosclerosis [17]. In diabetic nephropathy (DN), deletion of MALAT1 restrained pyroptosis in high-glucose-treated HK-2 cells [18,19], suggesting the regulatory functions of MALAT1 in pyroptosis signaling pathway. However, its potential roles and underlying in diabetic cerebral ischemia-related pyroptosis remain unknown. In the present study, we hypothesized that MALAT1 participated in the pathogenesis of the diabetic cerebral I/R injury via MALAT1/STAT1-mediated pyroptosis of neurons.

Animal model
Sixty db/db rats and forty BALB/c mice (weigh: 20±2g; sex: male; age: 8-10 weeks) were purchased from the Nanjing Medical University. The mice were raised in a dry and ventilated pathogen-free barrier facility with 60% relative humidity, 12h/d lighting time at 25°C with free access to food and water. After acclimatization for 2 days, mice were randomly divided into ve groups: normal group, cerebral I/R group (n=20; both using C57BL/6 mice), diabetes group, diabetic cerebral I/R group (n=20; both using db/db mice) and diabetic cerebral I/R+MALAT1 short hairpin RNA (sh-MALAT1) group (n=20; using db/db mice).
The mice in cerebral I/R group and diabetic cerebral I/R group were anesthetized using 1% pentobarbital sodium (50mg/kg), followed by performed to establish I/R model using middle cerebral artery occlusion (MCAO) method [21]. Brie y, mice were incised in the middle cervical region to expose and separate the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA).
Subsequently, the proximal part of CCA and the distal end of ECA were ligated. The opposite site of CCA was clamped. A bulletheaded mono lament nylon suture was inserted into the ECA until a slight resistance was obtained. After 2 h, the suture was removed to restore blood ow. The mice of the other two groups were modeled following the same procedure as above. At 36 or 72 h after reperfusion, neurological de cit score of each mouse was evaluated according to the criteria of Longa scoring standard [22]: 0-no neurological de cit symptoms; 1-unable to extend the left fore limb while lifting the tail; 2-circling toward the left side; 3-di cult to walk, fall toward the left side; 4-impaired in walk and unconsciousness. Whereafter, the mice were sacri ced by intraperitoneal injection of pentobarbital sodium (150mg/kg) and the brain tissues were resected for the subsequent experiment. The brain tissues were promptly stored in liquid nitrogen. All animal experiments were supervised by the Ethics Committee of First Clinical Hospital, Harbin Medical University.

The encephalaedema volume
After removing the lower brain stem and epencephalon, the brain tissue was sliced into 2mm-thickness slides. The slides were incubated with 2% TTC solution in the dark room at 37°C for 20 min. Next, 4% paraformaldehyde solution was used to x the slides for 24 h. The images were photographed and analyzed using Image J (ver. 1.37C). The volume of encephaledema was calculated according to the formula: V=∑(A1+A2)t/2. V: volume of encephaledema (mm 3 ); A1 and A2: front and back encephaledema area of the slides (mm 2 ); t: thickness of the slides (mm).

Cell hypoxia/reoxygenation (H/R) model
Cells from the mice brain tissues were isolated as previously described [24]. BV2 cell line was purchased form XX and recovered in our lab. Cells were divided into control group, high-glucose group, H/R group and high-glucose+H/R group. To establish the H/R cell model, the cells were cultured in glucose-free DMEM and incubated at 37°C in a multi-gas incubator with 94% N 2 , 5% CO 2 and 1% O 2 . Following hypoxia, Cells were incubated for another 4 h in standard DMEM at 37°C with 5% CO 2 for reoxygenation.

Lactate dehydrogenase (LDH) analysis
The amount of LDH was measured using a LDH Assay Kit (C0017; Beyotime) followed the manufacturer's manual. The medium was collected and LDH levels were detected. LDH amount was expressed as fold changes to the normal group.

ELISA
The brain tissues were homogenized and centrifuged for detecting the pro-in ammatory factors. The protein levels of IL-6 (K4144-100), TNF-α (K1051-100), and IL-1β (K4795-100; all from Biovision Inc.) were analyzed by the corresponding ELISA kits according to the manufacturer's protocols. The absorbance values were detected at 450 nm wavelength.

Flow cytometry assay
The cells were stained by SYTOX and caspase-1 (CA1020; Beijing Solarbio Science & Technology Co., Ltd.). Attune NxT Flow Cytometer and its supporting software (Thermo Fisher Inc.) was used for ow cytometry assay. 5 µl of SYTOX and anti-caspase-1 antibody were added to each well of the 6-well plate, and the cells were resuspended at the density of 1×10 6 ml. The results were detected using a ow cytometry.

Luciferase reporter assay
The wild and mutant types 3′-UTR region of STAT1 luciferase reporter vectors were designed and synthesized by Guangzhou RiboBio Co., Ltd. The cells were lysed to detect luciferase activities using the Co., Ltd.) following the manufacturer's protocol. Brie y, the cells were lysed and incubated with the biotinylated MALAT1 probe and its control probe. Streptavidin-labeled magnetic beads were resuspended and incubated with the probes (50 pmol) at 4°C overnight. Next, the beads were eluted from the RNAprotein complex. The proteins were resolved in SDS-PAGE and detected by western blotting assay. The MALAT1 primers sequences were: sense, 5′-CCATCGGCAAGACCAAGA-3′; anti-sense, 5′-ACAGGCTCAGAATGCTCAT C-3′.
2.14 RNA Binding Protein Immunoprecipitation (RIP) assay RIP assay was performed using the RNA Immunoprecipitation Kit (P0101; Geneseed) according to the manufacturer's protocol. Brie y, cells were resuspended in PBS at the density of 1×10 7 , then lysed on ice and centrifuged, after which 100 µl of supernatant was incubated with magnetic beads conjugated with human anti-STAT1 antibody (3472-30T) and negative control normal mouse IgG (6402-05; both from Biovision Inc.). The magnetic beads were eluted and RNA complex was puri ed to obtain the RNA for RT-qPCR analysis.

Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was conducted using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (cat no. 9003s; Cell Signaling Technology Inc.) followed the manufacturer's protocol. Brie y, cells were resuspended. Then, cells were crosslinked with 1% formaldehyde at 37˚C for 30 min. 0.125 M glycine was added to stop the reaction. Cells were lysed on ice and ultrasonic-treated to obtain the genomic DNA fragments. Subsequently, the fragments were coprecipitated with anti-STAT1 antibody (3472-30T). The speci c primers that were used to amplify the NLRP3 promoter DNA fragments. The DNA was puri ed for RT-qPCR analysis.
2.16 Fluorescence in situ hybridization (FISH) assay MALAT1 cellular expressions were analyzed using the Fluorescence In Situ Hybridization kit (F11201/50; Shanghai GenePharma Co., Ltd.) according to the manufacturer's protocol. Brie y, cells were inoculated into a 48-well plate and the medium was discarded. The cells were rinsed with PBS twice and then xed with 4% paraformaldehyde for 15 min. Thereafter, cells were hybridized with the Cyanine dye 5 (Cy5)labeled MALAT1 (designed and synthesized by GenePharma) probes at 37°C overnight. The next day, the probe mixture was discarded and the cells were counterstained with DAPI. The cells were observed and captured under a uorescence microscope.

Statistical analysis
The data were analyzed using SPSS 20.0, and represented as mean ± SD. Student t test was applied for analyzing the difference between two groups, and one-way ANOVA for the difference among multiple groups. P < 0.05 was deemed as statistical signi cance.

The pyroptosis of neurons was more remarkable in diabetic cerebral ischemia
The decrease of neurons was closely associated with the development of diabetic cerebral ischemia. As shown in Fig. 1A, the neurological de cit score of the I/R injury mice were signi cantly increased, which was more remarkable in db/db I/R group. The encephaledema volume of the I/R injury db/db mice was much larger than that of the I/R injury normal mice (Fig. 1B). This was consistent with the results from TTC staining. diabetic cerebral ischemia further promoted brain tissue damage ( Figure 1C). Moreover, the levels of proin ammatory cytokines, including TNF-α, IL-18 and IL-1β were measured. As shown in Fig. 1D-F, the levels of the pro-in ammatory cytokines was signi cantly increased in I/R group, especially in db/db I/R mice. Previous studies revealed that in ammatory-induced neural death (pyroptosis) is the key factor of cerebral I/R injury. We, thereafter, determined the expressions of pyroptosis biomarkers, NLRP3, ASC, caspase-1, and GSDMD. The protein expression of NLRP3, caspase-1 p20, and GSDMD-N was signi cantly increased in I/R group, which more potent in diabetic cerebral ischemia mice (Fig. 1G).

Knockdown of MALAT1 relieved cerebral I/R injury in diabetic mice
Through bioinformatic approaches, we found that several lncRNAs were aberrant expressed in diabetic cerebral ischemia ( Fig. 2A). We screened for seven upregulated lncRNAs, among which MALAT1 was most signi cantly upregulated (Fig. 2B). Therefore, MALAT1 was selected for the following study.

High glucose facilitated the pyroptosis of neurons induced by H/R in vitro
Cells were exposed to high glucose (HG) to mimic microglial cells in diabetic mice, and H/R was used to mimic I/R injury in vitro. As shown in Fig. 3A-D, H/R signi cantly increased cas-1/SYTOX positive cells as well as PI positive cells. Moreover, the increase of LDH level was further enhanced by HG (Fig. 3E). Additionally, HG increased the release of pro-in ammatory cytokyines, such as IL-18 and IL-1β ( Fig. 3F and G). GSDMD-N, caspase-1 p20 and NLRP3 expressions were facilitated in the H/R cells. HG signi cantly increased the expression of GSDMD-N, caspase-1 p20 and NLRP3 of H/R-treated cells.
Moreover, H/R treatment signi cantly increased the concentration of IL-18 and IL-1β, which was more potent in HG+H/R group ( Fig. 3H and I). HG facilitated the expression of MALAT1 (Fig. 3J).

Downregulating MALAT1 mitigated neural pyroptosis induced by diabetic cerebral ischemia in vitro
To futher verify the potential roles of MALAT1 in diabetic cerebral ischemia, we examined the effects of MALAT1 on diabetic cerebral ischemia model in vitro. As shown in Figure 4A, MALAT1 knockdown signi cantly decreased cas-1/SYTOX positive cells. Moreover, downregulation of MALAT1 signi cantly suppressed cytotoxicity (Fig. 4B) and in ammatory response ( Fig. 4C and D), manifested by the decrease in the release of LDH, IL-18 and IL-1β. Moreover, the protein expression levels of GSDMD-N, caspase-1 p20 and NLRP3 elevated by HG and H/R treatments was antagonized by MALAT1 knockdown (Fig. 4E).

MALAT1 positively regulated NLRP3 expression via binding to STAT1
Our preliminary experiment used Genome (http://genome.ucsc.edu/) to locate the promoter region of MALAT1. And ALGGEN (http://alggen.lsi.upc.es/) predicted that STAT1, a transcription factor, was able to bind to the promoter. We carried out RNA-pull down assay for validation. Anti-sense MALAT1 was used as a negative control. RNA pull-down and mass spectrometry analysis MALAT1 could interact with STAT1 ( Fig. 5A and B). Next, RIP assay further con rmed the interaction between MALAT1 and STAT1 (Fig. 5C). Cellular colocalization images showed that MALAT1 and STAT1 were mainly expressed in the cytoplasm, suggesting that MALAT1 may induce pyroptosis via interacting with STAT1 (Fig. 5D). Subsequently, the results of Genome and ALGGEN showed that STAT1 had binding sites with the promoter sequence of NLRP3. The sequences were predicted and proved by JASPAR (http://jaspar.genereg.net/) (Fig. 5E). This was further veri ed by luciferase activity. As shown in Figure 5F, knockdown of MALAT1 signi cantly decreased the luciferase activity in neuron cells (Fig. 5G). The mRNA and protein expression of NLRP3 was positively regulated by MALAT1 and STAT1 (Fig. 5H-J).
3.7 Knockdown of STAT1 reversed the effects of MALAT1 on cerebral injury of the diabetic I/R mice.
Sh-STAT1 and Ad-MALAT1 were injected into the diabetic I/R mice to further investigate the effects of STAT1 knockdown on diabetic cerebral ischemia in vivo. Sh-STAT1 decreased neurological de cit scores in diabetic I/R mice (Fig. 7A). Besides, injection of sh-STAT1 reduced the volume of encephaledema and suppressed brain tissues damages ( Fig. 7B and C). Moreover, STAT1 knockdown signi cantly suppressed the release of TNF-α, IL-18, and IL-1β ( Fig. 7D-F). The protein expression of pyroptosis biomarkers, including GSDMD-N, caspase-1 p20 and NLRP3, were signi cantly downregulated by sh-STAT1 ( Fig. 7G and H).

MALAT1 expression level was regulated by STAT1
We had elucidated that MALAT1 promoted the pyroptosis of neural cells via binding to STAT1. However, the mechanism how STAT1 regulated MALAT1 expression is still unknown. The mRNA expression of STAT1 was signi cantly increased in I/R+db/db mice (Fig. 8A). STAT1 was predicted to have three complementary sequences to MALAT1 (Fig. 8B). Moreover, STAT1 remarkable increased the luciferase activity in site 3, whereas there was no signi cant changes in site 1 and 2 ( Fig. 8C-E). RIP data showed that the MALAT1 enrichment of STAT1 was notably higher than that of IgG in site 3 (Fig. 8F). These data suggested that STAT1 targeted MALAT1 on site 3. Moreover, MALAT1 expression level was signi cantly reduced by STAT1 knockdown, but increased by STAT1 overexpression (Fig. 8G).

Discussion
In this study, we demonstrated that knockdown of MALAT1 relieved diabetic cerebral ischemia for the rst time. MALAT1 was upregulated in diabetic cerebral ischemia. However, knockdown of MALAT1 improved neurological de cits of the diabetic I/R mice model and ameliorated pyroptosis of neural cells. Mechanically, we found that MALAT1 interacted with the transcription factor STAT1 to induce the activation of NLRP3 in ammasome. Knockdown of MALAT1 inactivated NLRP3 signaling and suppressed the pyroptosis of neural cells and brain tissue damage induced by diabetic cerebral ischemia via interacting with STAT1. Moreover, STAT1 transcriptionally activated MALAT1. Hence, MALAT1 interacting with STAT1 promoted diabetic cerebral ischemia-induced pyroptosis through regulating NLRP3 signaling.
NLRP3 in ammasome, activated by various microbe-associated molecular patterns and damageassociated molecular patterns (DAMPs) induces the cascade of in ammatory response and damages brain tissues [8,25]. Pyroptosis, a pro-in ammatory pattern of death stimulated by in ammasome activation, is closely associated with neuron dysfunction and brain tissue damage. Pyroptosis is different from necrosis: 1) the activation of NLRP3 in ammasome [11][12][13]; 2) GSDMD mediated rupturing and blebbing of plasma membrane, which is the characteristics of pyroptosis [26]; 3) with canonical in ammasomes (engaging pro-caspase-1) and non-canonical in ammasomes (activating caspase-11) [27]. Diabetic cerebral ischemia is an autoimmune disease [28]. Diabetes mellitus suppresses white matter repair and promotes long-term cerebral ischemia injuries [29]. Diabetic cerebral ischemia stimulated NLRP3 in ammasomes (NLRP3, ASC and cas-1) and induced neuron dysfunction (in ammatory response and pyrotosis). Thence, to suppress the activation of NLRP3 may be a potential strategy for diabetic cerebral ischemia injuries.
LncRNAs are widely employed as targeting treatments for several diseases with the advantages of high speci city and mild side effects. It promoted high glucose-induced H9C2 cardiomyocyte pyroptosis via targeting miR-141-3p [30]. A previous study also revealed that MALAT1 accelerated high glucose-induced pyroptosis of endothelial cells partly by upregulating NLRP3 expression. Wang et al. [21] demonstrated that MALAT1 mediated the exacerbation of cerebral I/R injury induced by diabetes through triggering the in ammatory response in microglia via MyD88 signaling. Therefore, we hypothesized that knockdown of MALAT1 might relieve diabetic cerebral ischemia by inhibiting pyroptosis. We found that knockdown of MALAT1 ameliorated neurological de cits, encephaledema, and pyroptosis in vivo and restored neuron function in vitro. MALAT1 knockdown mediated inactivation of NLRP3 may protected against diabetic cerebral ischemia.
LncRNAs function in various pathological processes of human body through different mechanism, one of which is binding to speci c proteins. lncRNAs function as ceRNA to regulate gene expression via sponging microRNAs. lncRNAs also interacts with RNA binding protein to promote the mRNA stability of the target gene. Additionally, lncRNAs bind to transcription factors to transcriptionally regulate gene expression. For instance, Ni et al reveal that lncRNA GAS5 induces YAP phosphorylation, which further downregulates YTHDF3 suppresses m6A moti cation of GAS5 [31]. FOXN3-NEAT1-SIN3A forms a negative feedback loop to exacerbate the carcinogenesis of hormonally responsive breast cancer [32]. lncRNA uc.134 inhibits the aggressiveness of hepatocellular carcinoma via suppressing CUL4A-mediated ubiquitination of LATS1 [33]. In this study, lncRNA MALAT1 bound with STAT1 to induce the upregulation of NLRP3. STAT families determines immune responses in the microenvironment in tumor as well as in nerve disorders [34,35]. STAT1-mediated downregulation of N-methyl-D-aspartate receptors contributes to hippocampal neuron degeneration and memory de cits [36]. Inactivation of JAK/STAT1 signaling and IL-13 suppresses the pyroptosis of neurons and alleviates moderate traumatic brain injury [37]. In this study, STAT1 bound to the promoter region of NLRP3 and induce the in ammatory response in neuron cells, which nally increased the accumulation of pyrotosis executor GSDMD-N. Interestingly, MALAT1 knockdown suppressed the transcriptional activity of STAT1 and restore neuron cellular function. This may provide a new sight of diabetic cerebral ischemia.
We subsequently attempted to investigate the upstream regulatory mechanism of MALAT1 after we had demonstrated the MALAT1/STAT1/NLRP3 pathway. Interestingly, STAT1 was predicted to have three binding sites with MALAT1, and we proved that STAT1 regulated MALAT1 expression via the targeted relationship.
In conclusion, knockdown of lncRNA MALAT1 suppresses pyroptosis through inhibiting NLRP3 expression via STAT1, and MALAT1 is upstream regulated by STAT1 during diabetic cerebral ischemia. This study might contribute to a better understanding of diabetic cerebral ischemia pathogenesis and provide a basis for developing novel therapeutic strategies.

Declarations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Compliance with Ethical Standards
Yes.

Ethics approval
All human and animal experiments were supervised by the Ethics Committee of First Clinical Hospital, Harbin Medical University.

Consent to participate
Informed consent was obtained from all individual participants included in the study. All the participants were consent for publication.

Con icts of interest
The authors declared that they have no con ict interest.