LncRNA TUG1 contributes post-stroke NLRP3 inflammasome-dependent pyroptosis via miR-145a-5p/TLR4 axis

doi:10.21203/rs.3.rs-1512330/v1

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LncRNA TUG1 contributes post-stroke NLRP3 inflammasome-dependent pyroptosis via miR-145a-5p/TLR4 axis

https://doi.org/10.21203/rs.3.rs-1512330/v1

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Objective: Pyroptosis, a type of programmed cell death illuminated by inflammasomes and active caspases, is implicated in post-stroke inflammation. Our previous study showed that lncRNA taurine up-regulated gene 1 (TUG1) sponging miR-145a-5p modulated microglial activation after oxygen-glucose deprivation (OGD). However, the role and mechanism of TUG1 on post-stroke pyroptosis is not fully clear.

Methods: Photo-thrombosis stroke mice and OGD-treated BV-2 microglia were established respectively. TUG1 knockdown or overexpression was achieved by intraventricular infusion of AAV-shTUG1 in vivo, or transfection of siTUG1 and pcDNA3.1-TUG1 in vitro. Neurological function and infarction volume were evaluated. Meanwhile, pyroptosis-associated proteins (IL-1β, IL-18, NLRP3, ASC, cleaved-caspase-1 and GSDMD-N), TLR4 and p-p65/p65 as well as TUG1 and miR-145a-5p were detected 24 hours after photo-thrombosis or 4 hours after OGD by qRT-PCR, western blot and ELISA. The correlation between TUG1/miR-145a-5p/TLR4 axis and pyroptosis was explored by dual-luciferase reporter assay and functional gain-and-loss experiments.

Results: Photo-thrombosis or OGD caused neural injury and upregulated pyroptosis-associated proteins, TUG1, TLR4 and p-p65 as well as downregulated miR-145a-5p, which was prevented by TUG1 knockdown in vivo and in vitro. TLR4 gene, putatively binding with miR-145a-5p by bioinformatics analysis, was found to be a direct target of miR-145a-5p with negative interactions. Furthermore, miR-145a-5p inhibitor abolished the inhibitive effects of siTUG1 on TLR4 and p-p65 as well as pyroptosis-associated proteins, whereas miR-145a-5p mimics abrogated the enhanced effects of pcDNA3.1-TUG1 on that, suggesting an involvement of TUG1/miR-145a-5p/TLR4 axis on pyroptosis.

Conclusion: TUG1 contributes NLRP3 inflammasome-dependent pyroptosis through miR-145a-5p/TLR4 axis post stroke, providing a promising therapeutic strategy against inflammatory injury.

pyroptosis

TUG1

miR-145a-5p

TLR4

NLRP3 inflammasome

cerebral ischemia

Stroke is a leading cause of death and long-term disability worldwide, about 87% of which can be attributed to ischemic type[1, 2]. At present, thrombolysis within 4.5 hours and mechanical thrombectomy at most 24 hours are both approved therapies for acute ischemic stroke, nonetheless, the therapies are unpractical to stroke patients who are outside the time-window[3, 4]. Ischemic stroke generates sterile inflammation characterized by the activation of microglia and the release of inflammatory cytokines at the early stage, in which microglial pyroptosis plays an important role to cell death and brain injury[5-9]. Pyroptosis is a novel type of programmed cell death illuminated by inflammasomes and requires active caspases[10]. Typically, NOD-like receptor protein 3 (NLRP3) inflammasome-dependent pyroptosis is triggered by toll-like receptors (TLRs), a kind of pattern recognition receptors in respond to both pathogen-related molecule patterns and damage-related molecular patterns, and then facilitates the activation of caspase-1 (casp-1) and subsequent release of proinflammatory cytokines interleukins-1β (IL-1β) and interleukins-18 (IL-18)[11, 12]. Apparently, targeting the ischemia-induced pyroptosis would be beneficial to reduce inflammatory injury and improve stroke prognosis.

Long non-coding RNAs (LncRNAs), a type of non-protein-coding RNAs with more than 200 nucleotides, have been shown to modulate a large number of protein-coding genes and involve neural damage in numerous neurological diseases including stroke[13-15]. Our previous study found that lncRNA taurine up-regulated gene 1 (TUG1), which was highly expressed in BV-2 microglial cells, regulated microglial polarization and the production of inflammatory cytokines by sponging miR-145a-5p with negative interactions at the early stage after oxygen-glucose deprivation (OGD)[16]. Moreover, bioinformatics analysis reveals putative binding sites of miR-145a-5p with the 3’UTR region of TLR4, inferring that TUG1 may be implicated into pyroptosis through miR-145a-5p/TLR4 axis. The close correlation between pyroptosis and post-stroke inflammation urges us to conduct the present study focusing on the role and mechanism of TUG1 on NLRP3 inflammasome-dependent pyroptosis after stroke.

2.1 Photo-thrombosis (PT) stroke model and neurological evaluation

The animal experimental procedures were approved by the Ethics Committee of the Laboratory Animal Center of Sun Yat-sun University, Guangzhou, China, and were carried out in accordance with the ARRIVE guidelines. The animal was randomly divided into four group: sham control, PT group, PT+AAV-negative control (AAV-NC) and PT + AAV-shTUG1 group (n=18 per group). Anesthesia for all surgical procedures was achieved using an injection of 10% chloral hydrate (3 ml/kg, i.p.).

PT stroke model was established as previously reported[17]. In brief, adult male C57BL/6 mice were anesthetized and fixed on a stereotaxic instrument. An irradiation window was centered 1 mm lateral and right posterior to the bregma on the exposed skull. Immediately after an injection of photochemical dye Rose Bengal (20 mg/kg) (MP Biomedical, CA, USA) via the tail vein, the mice were illuminated with green light at 540 nm through the irradiation window at 2 mm diameter for 2 minutes. A laser speckle contrast imaging system (PeriCam PSI HD system, Stockholm, Sweden) was used to record the real-time changes of cortical blood flow in vivo. A laser probe was positioned about 12cm above the skull and the selected region of interest (ROI) area was 1 mm². We recorded 120 consecutive speckle images of mice for 2 minutes by a laser speckle contrast imaging system (PeriCam PSI HD, Stockholm, Sweden) . The real-time change of cortical blood flow at each time point in ROI was normalized and expressed as a relative value. The sham group was only illuminated without the injection of Rose Bengal. Throughout the procedure, respiratory status was in a smooth condition and body temperature was kept at 37.5°C with a heating pad.

Neurological function was blindly assessed in each group before and 24 hours after PT with a modified neurologic severity score (mNSS)[18]. The mNSS consists of a comprehensive test of motor, sensory, and reflex activities, with a score range of 0–14 points (1–4 as mild injury, 5–9 as moderate injury, 10–14 as severe injury).

2.2 TUG1 knockdown in vivo

Three weeks before PT, 2.5 μL of AAV-shTUG1 (1 × 10¹² v.g./mL, Gene Chem, Shanghai, China) was continuously infused into the lateral ventricle at a rate of 0.2 μl/min to knock down TUG1. The infusion was made via a microliter syringe (Hamilton Co., NV, USA) at the following stereotaxic coordinates: 0.3 mm posterior to the bregma, 1 mm right lateral to the midline, 2 mm beneath the surface of the skull. After the infusion was completed, the needle was kept in position for 15 minutes and then slowly pulled out. The wound was closed by bone cement and skin glue. AAV-NC (Gene Chem) was used as the control following the same operation.

2.3 Cortex tissue collection and staining

Twenty-four hours after PT or sham PT, 15 mice from each group were anesthetized and sacrificed. For immunofluorescence, the brains from six mice were perfused and post-fixed with 4% paraformaldehyde at 4℃ for 8 hours. Series of adjacent 10μm coronal frozen sections were collected at the infarcted level. For western blot and real-time quantitative PCR (qRT-PCR), the brains from the other six mice were perfused with pre-cooling heparinized saline. Subsequently, the peri-infarction cortex tissue was quickly obtained and stored at −80℃.

An anti-NeuN antibody (Abcam, Cambridge, UK) and an anti-Iba-1 antibody (WKAO, Tokyo, Japan) was used to mark cortical neurons and microglial cells, respectively. Sections were first incubated with the NeuN antibody or the Iba-1 antibody at 4℃ overnight followed by species-specific Alexa Fluor plus 555-or 488-conjugated antibody (Thermo Fisher Scientific, MA, USA) at room temperature for 1 hour. Thereafter, sections were counterstained with DAPI and analyzed under a fluorescence microscope (Nikon DS-Ri2, Japan). To observe the gross infarcted zone, small 2mm coronal blocks from the brain were stained with 2,3,5-Triphenyltetrazolium chloride (TTC, Solarbio, Beijing, China) at room temperature for 30 minutes, then photographed and calculated using the ImageJ software (National Institutes of Health, MD, USA).

2.4 Cell culture and OGD

BV-2 microglial cells (Kunming Cell Bank, Chinese Academy of Sciences) were cultured in Dulbecco’s modified eagle medium (Gibco, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific) and 1% antibiotics (TransGen Biotech, Beijing, China) in a humidified incubator under 5% CO₂ at 37℃. Cells were split at 70%–80% confluence. OGD was induced to mimic ischemic conditions in vitro[16]. Briefly, cells were cultured in glucose-free Dulbecco’s modified eagle medium, and flushed with 95% N2/5% CO2 gas mixture at a flow rate of 4 l/min for 10 minutes to create an anaerobic condition. A gas analyzer (Coy Laboratory, MI, USA) was used to monitor the anaerobic condition. Cells were then transferred to normal culture medium under 5% CO2 for reoxygenation. Our previous study showed that the regimen of OGD for 4 hours and reoxygenation for 24 hours was able to induce pivotal signaling events in cells without causing excessive death[16]. Control cells were treated without OGD.

2.5 Cell transfection

TUG1 small interfering RNA (siTUG1), pcDNA3.1-TUG1 vector, miR-145a-5p mimics, miR-145a-5p inhibitor as well as their NCs were constructed by Tsigke (Guangzhou, China) and GenePharma (Guangzhou, China), respectively. The corresponding sequences are listed in Table 1. A lipofectamine 3000 reagent kit (Invitrogen, CA, USA) was used to transfect the cells (3´10⁶/well) in a 6-well plate according to the manufacturer’s protocols. The amount of transfection was 2.5μg for pcDNA3.1-TUG1 vector and its control, or 150 nmol for the others. Transfected cells were incubated 24 hours and then subjected to OGD treatment. The efficiency of transfection was confirmed by qRT-PCR.

Table 1 Nucleotide sequences used in the study

Name	Primer	Sequences
siTUG1	Sense	5’-CCAUCUCACAAGGCUUCAATT-3’
	Anti-sense	5’-UUGAAGCCUUGUGAGAUGGTT-3’
siTUG1 NC	Sense	5'-UUCUCCGAACGUUGUCACGUTT-3'
	Anti-sense	5'-TTAAGAGGCUUGCACAGUGCA-3'
mmu-miR-145a-5p mimics	Sense	5'-GUCCAGUUUUCCCAGGAAUCCCU
	Anti-sense	5'-GGAUUCCUGGGAAAACUGGACUU-3'
mimics NC	Sense	5'-UUCUCCGAACGUGUCACGUTT-3'
	Anti-sense	5'-ACGUGACACGUUCGGAGAATT-3'
mmu-miR-145a-5p inhibitor		5'-AGGGAUUCCUGGGAAAACUGGAC-3'
inhibitor NC		5'-CAGUACUUUUGUGUAGUACAA-3'
GAPDH	Forward	5’-TGCCCAATTCCACCAAGGAA-3’
	Reverse	5’-CTGCCAACCTTCTATACGCCT-3’
TUG1	Forward	5'-CAAACCTGGCTCTACAATCCTATT-3'
	Reverse	5'-CATTCAGCAATCAGGAGGCAC-3'
U6	Forward	5'-CTCGCTTCGGCAGCACATATACT-3'
	Reverse	5'-ACGCTTCACGAATTTGCGTGTC-3'
miR-145a-5p	miR-145a-5p RT	5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGATTCC -3'
	Forward	5'-ACACTCCAGCTGGGGTCCAGTTTTCCCAGG -3'
	Reverse	5'-GGTGTCGTGGAGTCG -3'

2.6 qRT-PCR

Total RNAs were extracted from BV-2 cells or peri-infarction cortex tissue using a RNAzol RT reagent (MRC, OH, USA), followed by the reverse-transcription to cDNA using a PrimeScript™ RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. The relative level of TUG1 or miR-145a-5p to GAPDH or U6 was quantitatively measured in 2^-ΔΔCt method using a TB Green® Premix Ex Taq™ kit (Takara) on ABI 7,500 real-time system (Applied Biosystems, CA, USA). The primer sequences were synthesized by Tsigke (Guangzhou) and are listed in Table 1. All samples were performed at least three parallel reactions.

2.7 Western blot

Proteins were extracted from peri-infarction cortex tissue or cultured cells using RIPA lysis buffer plus 10 µl/mL protease inhibitor (Thermo Fisher Scientific). The protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific). Equivalent amount of proteins from each sample was resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Blots were incubated at 4℃ overnight with the following primary antibodies: TLR4, p-p65, p65, gasdermin D (GSDMD), apoptosis-associated speck-like protein containing CARD (ASC), IL-1β, IL-18, and GAPDH (all 1:1000, Affinity Biosciences, OH, USA); NLRP3 (1:1000, Abcam); casp-1 (1:1000, AdipoGen, Liestal, Switzerland). Antibodies binding to blots were visualized using an HRP-linked secondary antibody kit (1:3000, CST, MA, USA). The relative levels of proteins to GAPDH were measured using ImageJ software.

2.8 ELISA

Blood samples from the ocular vein of mice and cultured supernatant from BV-2 cells in different treatment groups were carefully collected and centrifuged to remove cell debris and concretionary stuff. The concentration of IL-1β and IL-18 in the serum and the cultured supernatant were quantified using specific ELISA kits (Elabscience, Wuhan, China) following the manufacturer’s instructions. A standard curve by using a standard solution was made to normalize the measurement.

2.9 Dual luciferase reporter gene assay

The putative binding sites between miR-145a-5p and TLR4 are predicted by searching an online database (http://www.targetscan.org). The 3’UTRs of both wild type and mutated TLR4 (TLR4 3’-UTR-WT or 3’-UTR-MUT) were constructed by Tsigke (Guangzhou), and separately inserted into the pmirGLO vector (Promega, Madison, USA). The TLR4 3’-UTR-WT or 3’-UTR-MUT vector, as well as miR-145a-5p mimics or mimics NC were co-transfected into BV-2 cells using a lipofectamine 3000 reagent kit (Invitrogen). Transfected cells were harvested 48 hours later and the luciferase activity was detected using a dual-luciferase reporter assay kit (TransGen Biotech, Beijing, China) according to the manufacturer's instructions.

2.10 Statistical analysis

All data were analyzed with GraphPad Prism 8.0 software (GraphPad Inc., CA, USA) in a blinded manner without knowledge of the treatment assignment. Numerical data were presented as the mean ± standard deviation. Student’s t-test or a general linear model with Bonferroni correction for analysis of variance was used to detect any intergroup differences when appropriate. A two-tailed P-value of 0.05 or less inferred statistical significance.

3.1 PT resulted in local cortex infarction with upregulated TUG1 and downregulated miR-145a-5p, relieved by TUG1 knockdown

PT conducted a consistent infarction in the right cerebral cortex, shown by decreased local cortical blood flow using laser speckle imaging and TTC staining (Fig.1A and B, P<0.001). In the meantime, decreased number of NeuN-positive neurons and increased Iba-1-positive microglia in the peri-infarction cortex as well as impaired neurological function were observed 24 hours after PT (Fig.1C and D, P<0.05). When compared with vehicle treatment, TUG1 knockdown by intraventricular infusion of AAV-shTUG1 salvaged cortex neurons, reduced microglia accumulation and infarct volume with improved neurological function (Fig 1B-D, P<0.05), demonstrating a detrimental role of TUG1 to neuronal survival post stroke. In addition, the levels of TUG1 and miR-145a-5p were measured by RT-qPCR. TUG1 was upregulated while miR-145a-5p was downregulated 24 hours after PT. By contrast, AAV-shTUG1 infusion obviously lowered TUG1 and raised miR-145a-5p, implying the role of TUG1 could be probably through suppressing miR-145a-5p (Fig.1E, P<0.01). The level of TUG1 was effectively knocked down by AAV-shTUG1 was shown in Supplementary Fig.S1.

3.2 TUG1 knockdown inhibited ischemia-induced pyroptosis in vivo and in vitro

The in vivo activity of pyroptosis was evaluated 24 hours after PT by detecting pyroptosis-associated proteins in both the serum and the ipsilateral cortex using ELISA and western blot. The protein levels of IL-1β, IL-18, NLRP3, ASC, cleaved-casp-1 and GSDMD-N were notably increased. TUG1 knockdown by AAV-shTUG1 intraventricular infusion reduced the levels of these proteins compared with the vehicle treatment. The differences were statistically significant (Fig.2A and B, P<0.05). Similarly, the levels of all these pyroptosis-associated proteins were also enhanced in OGD-treated BV-2 cells in vitro, together with upregulated TUG1 and downregulated miR-145a-5p. The changes after OGD treatment were sufficiently suppressed by siTUG1 transfection, suggesting that TUG1 could promote ischemia-induced pyroptosis (Fig.3A-E, P<0.05).

3.3 TLR4 gene was a direct target of miR-145a-5p

Online bioinformatics analysis reveals putative binding sites of the 3'UTR region of TLR4 with miR-145a-5p (Fig.4A). The direct binding between them was further confirmed in the present study. After the verification of miR-145a-5p mimics increasing miR-145a-5p while miR-145a-5p inhibitor decreasing miR-145a-5p in vitro (Fig.4B and C, P<0.01), the targeting relationship between 3′UTR region of TLR4 and miR-145a-5p was detailly explored by constructing TLR4 3′UTR-WT and 3′UTR-MUT reporter vectors. Dual luciferase reporter gene assay showed that miR-145a-5p mimics significantly lessened the relative luciferase activity of WT-TLR4 reporter gene but had no significant effect on the activity of MUT-TLR4 reporter gene (Fig.4D, P<0.01). Furthermore, miR-145a-5p mimics reduced the protein level of TLR4 while miR-145a-5p inhibitor raised that of TLR4 in BV-2 cells by western blot (Fig 4E and F, P<0.05), exhibiting that miR-145a-5p was a negative regulator directly targeting to TLR4 gene.

3.4 TUG1 contributed ischemia-induced pyroptosis through miR-145a-5p/TLR4 axis

Our previous study found that TUG1 sponged miR-145a-5p with negative interactions[16]. In the present study, the levels of TLR4 and p-p65 were distinctly incremental 24 hours after PT and 4-hour OGD. By contrast, TUG1 knockdown by using AAV-shTUG1 or siTUG1 significantly lowered the levels of TLR4 and p-p65 in vivo and in vitro (Fig.5A-C, P<0.05). We further explored whether TUG1 regulated ischemia-induced pyroptosis through miR-145a-5p/TLR4 axis in OGD-treated BV-2 cells by functional gain-and-loss experiments. TUG1 knockdown or overexpression was efficiently achieved by siTUG1 or pcDNA3.1-TUG1 transfection (Fig.5D, P<0.01). Western blot analysis showed that siTUG1 inhibited the levels of TLR4 and p-p65 as well as pyroptosis-associated proteins (IL-18, IL-1β, NLRP3, ASC, cleaved-caspase-1 and GSDMD-N), which was turned over by miR-145a-5p inhibitor. In comparison, pcDNA3.1-TUG1 increased the levels of TLR4 and p-p65 as well as pyroptosis-associated proteins, which was reversed by miR-145a-5p mimics (Fig.5E, P<0.05). Likewise, ELISA results also indicated that siTUG1-induced downregulation of IL-18 and IL-1β was abolished by miR-145a-5p inhibitor, whereas pcDNA3.1-TUG1-induced upregulation of that was abrogated by miR-145a-5p mimics (Fig.5F, P<0.05), indicating that pyroptosis was regulated by TUG1/miR-145a-5p/TLR4 axis involving NF-κb signaling.

Pyroptosis is morphologically characterized by cell swelling and eventually membrane rupture, distinctly different from other programmed cell deaths[19, 20]. In the canonical inflammasome pathway of pyroptosis, NLRP3 inflammasome, which is composed of NLRP3, ASC and pro-casp-1, is assembled from the recognition of disadvantaged stimulation by TLRs[21-23]. Upon the activation of casp-1, pro-IL-1β and pro-IL-18 are cleaved to their mature forms and GSDMD is to its GSDMD-N form. GSDMD-N binds to the plasma membrane and forms open pores across the plasma membrane, causing the release of mature IL-1β and IL-18 through the membrane pores into the extracellular matrix with a result of pyroptotic inflammation[24, 25]. In the present study, we observed Iba-1-positive microglia was accumulated in the peri-infarction zone at 24 hours after PT. It is believed for now that microglial pyroptosis is an important mediator of the innate immune response to ischemic injury and are considered as markers of neuroinflammation [26-30]. After ischemic stroke, all or part of these pyroptosis-associated proteins were upregulated in microglia at the early stage, showing enhanced pyroptosis activity[31, 32]. We also observed Iba-1 positive microglia specifically accumulated in the cortex of peri-infarct zone at 24h after PT. Meanwhile, NLRP3 knockout markedly reduced infarction volume and preserved permeability of blood-brain barrier with improved neurological function, indicative of a potential therapeutic strategy for stroke by inhibiting pyroptosis[33, 34]. Previous experimental studies demonstrated that TLR4/NF-κb was an important signaling pathway to reinforce NLRP3 inflammasome-dependent pyroptosis after ischemia[35], nonetheless, the upstream regulator on TLR4/NF-κb pathway need to be further clarified.

LncRNAs have been found to modulate gene expressions at epigenetic, transcriptional, and post-transcriptional levels, in which the lncRNA-miRNA-mRNA ceRNA network is implicated in multiple biological processes and development of diseases[36, 37]. TUG1 was originally thought to be an oncogenic lncRNA, an upregulation of which closely contributed to tumorigenesis[38, 39]. Recent studies revealed that TUG1 was abnormally upregulated after ischemic injury in various tissue, inhibiting the expression of antioxidant genes and participating in post-ischemia inflammation[40, 41]. Our team also found that TUG1, acting as a ceRNA sponging miR145a-5p with negative interactions, strengthened microglial polarization and inflammatory response after OGD[16]. Nevertheless, the role and mechanism of TUG1 on post-stroke pyroptosis is not fully clear. By using in vivo PT stroke model, we observed that TUG1 was upregulated while miR-145a-5p was downregulated in the ipsilateral cortex 24 hours after PT, in parallel with impaired neurological function and lessened cortex neurons. Meanwhile, the activity of pyroptosis was elevated, represented by higher levels of pyroptosis-associated proteins (IL-1β, IL-18, NLRP3, ASC, cleaved-casp-1 and GSDMD-N). It can be deduced that TUG1 and miR-145a-5p was drawn into post-stroke pyroptosis. Indeed, we further found that TUG1 knockdown by AAV-shTUG1 infusion increased the level of miR-145a-5p and the number of cortical neurons, simultaneously, decreased the levels of these pyroptosis-associated proteins with better neurological function. All above post-stroke changes were also reconfirmed in OGD-treated BV-2 cells in vitro. Altogether, in the context of TUG1 directly sponging miR145a-5p, the data indicated that TUG1 contributed to post-stroke pyroptosis through miR-145a-5p.

MiR-145 has been proved to function as an inflammatory regulator of cell survival by specifically targeting specific gene transcription[42]. On condition of a predicted correlation between miR-145a-5p and TLR4 by bioinformatics analysis, we confirmed that miR-145a-5p was a negative regulator directly binding to TLR4 gene by dual luciferase reporter gene assay in the present study. With the administration of AAV-shTUG1 or siTUG1, the elevation of TLR4 and p-p65 induced by PT and OGD was inhibited. Thus, it is reasonable to speculate that TUG1/miR-145a-5p could guide post-stroke pyroptosis ignited by TLR4/NF-κb. For further verification, functional gain-and-loss experiments were performed in OGD-treated BV-2 cells and the results showed that miR-145a-5p inhibitor prevented the inhibitive effects of siTUG1 to pyroptosis-associated proteins and TLR4/p-p65, whereas miR-145a-5p mimics abrogated the positive effects of pcDNA3.1-TUG1 to that. Taken together, the data demonstrated that TUG1/miR-145a-5p/TLR4 axis facilitated ischemia-induced pyroptosis involving NF-κb signaling.

There were several limitations in our study. First, we used BV-2 microglia as a substitute for primary microglia. Primary microglia are undoubtedly the best candidate for microglial study. However, BV-2 microglia have also been widely used in many microglial studies due to the most reservation of major characteristics and function of primary microglia[43, 44]. Second, pyroptosis can be induced by non-canonical inflammasome pathway, in which caspase-4/5/11 straightly cleave GSDMD excluding assembly of inflammasome. We did not detect the levels of these caspases, and cannot evaluate the role of TUG1 on non-canonical inflammasome pathway. Future studies are needed to clarify these issues

In conclusion, the present study supports the results that lncRNA TUG1 contributes NLRP3 inflammasome-dependent pyroptosis through miR-145a-5p/TLR4 axis involving NF-κb signaling post stroke (Fig.6), providing a promising therapeutic strategy against inflammatory injury.

Funding

This work was supported by grants from the National Natural Scientific Foundation of Guangdong, China (No. 2019A1515011600), the National Natural Scientific Foundation of China (No. 82071366), the Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (No. 2015B050501003).

Conflict of Interest

None of the authors have a conflict of interest to declare.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Meiling Yao, Ying Luo and Hongjie Li. The first draft of the manuscript was written by Meiling Yao. Songjie Liao and Jian Yu directed the study and revised the manuscript. All authors read and approved the final manuscript.

Data availability

All data for this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

All experimental procedures were approved by the Ethics Committee for Institutional Animal Care and Use Committee of Sun Yat-Sen University (NO: SYSU-IACUC-2020-000229).

Conflict of Interest

None of the authors have a conflict of interest to declare.

Consent to Participate

Not applicable.

Consent for publication

Not applicable.

Patel P, Yavagal D, Khandelwal P (2020) Hyperacute Management of Ischemic Strokes: JACC Focus Seminar. J Am Coll Cardiol 75(15):1844-1856. https://10.1016/j.jacc.2020.03.006
Verhoeven JI, Allach Y, Vaartjes I, Klijn C, de Leeuw FE (2021) Ambient air pollution and the risk of ischaemic and haemorrhagic stroke. Lancet Planet Health 5(8):e542-e552. https://10.1016/S2542-5196(21)00145-5
Duan Q, Sun W, Yuan H, Mu X (2018) MicroRNA-135b-5p prevents oxygen-glucose deprivation and reoxygenation-induced neuronal injury through regulation of the GSK-3beta/Nrf2/ARE signaling pathway. Arch Med Sci 14(4):735-744. https://10.5114/aoms.2017.71076
Dong X, Gao J, Zhang CY, Hayworth C, Frank M, Wang Z (2019) Neutrophil Membrane-Derived Nanovesicles Alleviate Inflammation To Protect Mouse Brain Injury from Ischemic Stroke. Acs Nano 13(2):1272-1283. https://10.1021/acsnano.8b06572
Kronenberg G, Uhlemann R, Richter N, Klempin F, Wegner S, Staerck L, Wolf S, Uckert W et al (2018) Distinguishing features of microglia- and monocyte-derived macrophages after stroke. Acta Neuropathol 135(4):551-568. https://10.1007/s00401-017-1795-6
Lee SW, de Rivero VJ, Truettner JS, Dietrich WD, Keane RW (2019) The role of microglial inflammasome activation in pyroptotic cell death following penetrating traumatic brain injury. J Neuroinflammation 16(1):27. https://10.1186/s12974-019-1423-6
Li J, Hao J, Yao D, Li R, Li X, Yu Z, Luo X, Liu X et al (2020) Caspase-1 inhibition prevents neuronal death by targeting the canonical inflammasome pathway of pyroptosis in a murine model of cerebral ischemia. Cns Neurosci Ther 26(9):925-939. https://https://doi.org/10.1111/cns.13384
Lee SW, Gajavelli S, Spurlock MS, Andreoni C, de Rivero VJ, Bullock MR, Keane RW, Dietrich WD (2018) Microglial Inflammasome Activation in Penetrating Ballistic-Like Brain Injury. J Neurotrauma 35(14):1681-1693. https://10.1089/neu.2017.5530
Serdar M, Kempe K, Rizazad M, Herz J, Bendix I, Felderhoff-Muser U, Sabir H (2019) Early Pro-inflammatory Microglia Activation After Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats. Front Cell Neurosci 13:237. https://10.3389/fncel.2019.00237
Kesavardhana S, Malireddi R, Kanneganti TD (2020) Caspases in Cell Death, Inflammation, and Pyroptosis. Annu Rev Immunol 38:567-595. https://10.1146/annurev-immunol-073119-095439
Goulopoulou S, Mccarthy CG, Webb RC (2016) Toll-like Receptors in the Vascular System: Sensing the Dangers Within. Pharmacol Rev 68(1):142-167. https://10.1124/pr.114.010090
Moraes-Vieira PM, Yore MM, Sontheimer-Phelps A, Castoldi A, Norseen J, Aryal P, Simonyte SK, Kahn BB (2020) Retinol binding protein 4 primes the NLRP3 inflammasome by signaling through Toll-like receptors 2 and 4. Proc Natl Acad Sci U S A 117(49):31309-31318. https://10.1073/pnas.2013877117
Boon RA, Jae N, Holdt L, Dimmeler S (2016) Long Noncoding RNAs: From Clinical Genetics to Therapeutic Targets? J Am Coll Cardiol 67(10):1214-1226. https://10.1016/j.jacc.2015.12.051
Yu S, Yu M, He X, Wen L, Bu Z, Feng J (2019) KCNQ1OT1 promotes autophagy by regulating miR-200a/FOXO3/ATG7 pathway in cerebral ischemic stroke. Aging Cell 18(3):e12940. https://10.1111/acel.12940
Wang C, Dong J, Sun J, Huang S, Wu F, Zhang X, Pang D, Fu Y et al (2021) Silencing of lncRNA XIST impairs angiogenesis and exacerbates cerebral vascular injury after ischemic stroke. Mol Ther Nucleic Acids 26:148-160. https://10.1016/j.omtn.2021.06.025
Wang H, Liao S, Li H, Chen Y, Yu J (2019) Long Non-coding RNA TUG1 Sponges Mir-145a-5p to Regulate Microglial Polarization After Oxygen-Glucose Deprivation. Front Mol Neurosci 12:215. https://10.3389/fnmol.2019.00215
Dietrich WD, Watson BD, Busto R, Ginsberg MD, Bethea JR (1987) Photochemically induced cerebral infarction. I. Early microvascular alterations. Acta Neuropathol 72(4):315-325. https://10.1007/BF00687262
Li Y, Chopp M, Chen J, Wang L, Gautam SC, Xu YX, Zhang Z (2000) Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab 20(9):1311-1319. https://10.1097/00004647-200009000-00006
Kovacs SB, Miao EA (2017) Gasdermins: Effectors of Pyroptosis. Trends Cell Biol 27(9):673-684. https://10.1016/j.tcb.2017.05.005
Wang M, Jiang S, Zhang Y, Li P, Wang K (2019) The Multifaceted Roles of Pyroptotic Cell Death Pathways in Cancer. Cancers (Basel) 11(9). https://10.3390/cancers11091313
Rathinam VA, Fitzgerald KA (2016) Inflammasome Complexes: Emerging Mechanisms and Effector Functions. Cell 165(4):792-800. https://10.1016/j.cell.2016.03.046
Shin S, Brodsky IE (2015) The inflammasome: Learning from bacterial evasion strategies. Semin Immunol 27(2):102-110. https://10.1016/j.smim.2015.03.006
Nystrom S, Antoine DJ, Lundback P, Lock JG, Nita AF, Hogstrand K, Grandien A, Erlandsson-Harris H et al (2013) TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis. Embo J 32(1):86-99. https://10.1038/emboj.2012.328
Wang L, Hauenstein AV (2020) The NLRP3 inflammasome: Mechanism of action, role in disease and therapies. Mol Aspects Med 76:100889. https://10.1016/j.mam.2020.100889
He WT, Wan H, Hu L, Chen P, Wang X, Huang Z, Yang ZH, Zhong CQ et al (2015) Gasdermin D is an executor of pyroptosis and required for interleukin-1beta secretion. Cell Res 25(12):1285-1298. https://10.1038/cr.2015.139
Qin X, Sun ZQ, Dai XJ, Mao SS, Zhang JL, Jia MX, Zhang YM (2012) Toll-like receptor 4 signaling is involved in PACAP-induced neuroprotection in BV2 microglial cells under OGD/reoxygenation. Neurol Res 34(4):379-389. https://10.1179/1743132812Y.0000000028
Wang X, Chen S, Ni J, Cheng J, Jia J, Zhen X (2018) miRNA-3473b contributes to neuroinflammation following cerebral ischemia. Cell Death Dis 9(1):11. https://10.1038/s41419-017-0014-7
Xu S, Wang J, Zhong J, Shao M, Jiang J, Song J, Zhu W, Zhang F et al (2021) CD73 alleviates GSDMD-mediated microglia pyroptosis in spinal cord injury through PI3K/AKT/Foxo1 signaling. Clin Transl Med 11(1):e269. https://10.1002/ctm2.269
Poh L, Kang SW, Baik SH, Ng G, She DT, Balaganapathy P, Dheen ST, Magnus T et al (2019) Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke. Brain Behav Immun 75:34-47. https://10.1016/j.bbi.2018.09.001
Cai M, Zhuang W, Lv E, Liu Z, Wang Y, Zhang W, Fu W (2022) Kaemperfol alleviates pyroptosis and microglia-mediated neuroinflammation in Parkinson's disease via inhibiting p38MAPK/NF-kappaB signaling pathway. Neurochem Int 152:105221. https://10.1016/j.neuint.2021.105221
Poh L, Kang SW, Baik SH, Ng G, She DT, Balaganapathy P, Dheen ST, Magnus T et al (2019) Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke. Brain Behav Immun 75:34-47. https://10.1016/j.bbi.2018.09.001
Yang F, Wang Z, Wei X, Han H, Meng X, Zhang Y, Shi W, Li F et al (2014) NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke. J Cereb Blood Flow Metab 34(4):660-667. https://10.1038/jcbfm.2013.242
Yang F, Wang Z, Wei X, Han H, Meng X, Zhang Y, Shi W, Li F et al (2014) NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke. J Cereb Blood Flow Metab 34(4):660-667. https://10.1038/jcbfm.2013.242
Feng YS, Tan ZX, Wang MM, Xing Y, Dong F, Zhang F (2020) Inhibition of NLRP3 Inflammasome: A Prospective Target for the Treatment of Ischemic Stroke. Front Cell Neurosci 14:155. https://10.3389/fncel.2020.00155
Chen J, Yang C, Xu X, Yang Y, Xu B (2018) The effect of focal cerebral ischemia-reperfusion injury on TLR4 and NF-kappaB signaling pathway. Exp Ther Med 15(1):897-903. https://10.3892/etm.2017.5463
Bao MH, Szeto V, Yang BB, Zhu SZ, Sun HS, Feng ZP (2018) Long non-coding RNAs in ischemic stroke. Cell Death Dis 9(3):281. https://10.1038/s41419-018-0282-x
Frank F, Kavousi N, Bountali A, Dammer EB, Mourtada-Maarabouni M, Ortlund EA (2020) The lncRNA Growth Arrest Specific 5 Regulates Cell Survival via Distinct Structural Modules with Independent Functions. Cell Rep 32(3):107933. https://10.1016/j.celrep.2020.107933
Wang P, Yang Z, Ye T, Shao F, Li J, Sun N, He J (2020) lncTUG1/miR-144-3p affect the radiosensitivity of esophageal squamous cell carcinoma by competitively regulating c-MET. J Exp Clin Cancer Res 39(1):7. https://10.1186/s13046-019-1519-y
Li Y, Zheng D, Pan L, Dai Y, Cai S, Zhao L, Zhu H (2019) Knockdown of TUG1 by shRNA inhibited renal cell carcinoma formation by miR-299-3p/VEGF axis in vitro and in vivo. Eur J Pharmacol 860:172536. https://10.1016/j.ejphar.2019.172536
Chen S, Wang M, Yang H, Mao L, He Q, Jin H, Ye ZM, Luo XY et al (2017) LncRNA TUG1 sponges microRNA-9 to promote neurons apoptosis by up-regulated Bcl2l11 under ischemia. Biochem Biophys Res Commun 485(1):167-173. https://10.1016/j.bbrc.2017.02.043
Wu X, Liu Y, Mo S, Wei W, Ye Z, Su Q (2021) LncRNA TUG1 competitively binds to miR-340 to accelerate myocardial ischemia-reperfusion injury. Faseb J 35(1):e21163. https://10.1096/fj.202000827RR
Vacante F, Denby L, Sluimer JC, Baker AH (2019) The function of miR-143, miR-145 and the MiR-143 host gene in cardiovascular development and disease. Vascul Pharmacol 112:24-30. https://10.1016/j.vph.2018.11.006
Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M (2009) The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX 26(2):83-94. https://10.14573/altex.2009.2.83
Chantong B, Kratschmar DV, Nashev LG, Balazs Z, Odermatt A (2012) Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells. J Neuroinflammation 9:260. https://10.1186/1742-2094-9-260

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LncRNA TUG1 contributes post-stroke NLRP3 inflammasome-dependent pyroptosis via miR-145a-5p/TLR4 axis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Photo-thrombosis (PT) stroke model and neurological evaluation

2.2 TUG1 knockdown in vivo

2.3 Cortex tissue collection and staining

2.4 Cell culture and OGD

2.5 Cell transfection

2.6 qRT-PCR

2.7 Western blot

2.8 ELISA

2.9 Dual luciferase reporter gene assay

2.10 Statistical analysis

3. Results

3.1 PT resulted in local cortex infarction with upregulated TUG1 and downregulated miR-145a-5p, relieved by TUG1 knockdown

3.2 TUG1 knockdown inhibited ischemia-induced pyroptosis in vivo and in vitro

3.3 TLR4 gene was a direct target of miR-145a-5p

3.4 TUG1 contributed ischemia-induced pyroptosis through miR-145a-5p/TLR4 axis

4. DISCUSSION

5. Conclusion

Declarations

References

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