Silencing of microRNA‐494 inhibits the neurotoxic Th1 shift via regulating HDAC2‐STAT4 cascade in ischaemic stroke

Abstract

Background and Purpose T helper cell 1 (Th1)‐skewed neurotoxicity contributes to the poor outcome of stroke in rodents. Here, we have elucidated the mechanism of the Th1/Th2 shift in acute ischaemic stroke (AIS) patients at hyperacute phase and have looked for a miRNA‐based therapeutic target. Experimental Approach MiR‐494 levels in blood from AIS patients and controls were measured by real‐time PCR. C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and cortical neurons were subjected to oxygen–glucose deprivation. Luciferase reporter system, chromatin immunoprecipitation sequencing (ChIP‐Seq), and ChIP‐PCR were used to uncover possible mechanisms. Key Results In lymphocytes from AIS patients, there was a Th1/Th2 shift and histone deacetylase 2 (HDAC2) was markedly down‐regulated. ChIP‐seq showed that HDAC2 binding sites were enriched in regulation of Th1 cytokine production, and ChIP‐PCR confirmed that HDAC2 binding was changed at the intron of STAT4 and the promoter of T‐box transcription factor 21 (T‐bet) in lymphocytes from AIS patients. MiR‐494 was the most significantly increased miRNA in lymphocytes from AIS patients, and miR‐494‐3p directly targeted HDAC2. A strong association existed between miR‐494 and Th1 cytokines, and neurological deficit as measured by the National Institute of Health Stroke Scale (NIHSS) in AIS patients. In vitro and in vivo experiments showed that antagomir‐494 reduced Th1 shift‐mediated neuronal and sensorimotor functional damage in the mouse model of ischaemic stroke, via the HDAC2‐STAT4 pathway. Conclusion and Implications We demonstrated that miR‐494 inhibition prevented Th1‐skewed neurotoxicity through regulation of the HDAC2‐STAT4 cascade.

miRNA-Coordinated Schizophrenia Risk Network Cross-Talk With Cardiovascular Repair and Opposed Gliomagenesis

“…Importantly, in the case of the latter miRNA, protection against the development of the tumors comes at a cost of elevated susceptibility to neurotoxicity, after exposure to ischemia/ reperfusion for example. Notably, knockdown of hsa-miR-494-5p reverses the neurotoxic phenotype in multiple models (Song et al, 2017;Deng et al, 2019;Zhao et al, 2019a;Zhao et al, 2019b). Hsa-miR-494-5p-dependent antagonistic relationships between gliomagenesis and neurotoxicity are intriguing, as they support previously noted decrease in odds of the development of brain tumors in patients with schizophrenia (Grinshpoon et al, 2005;Levav et al, 2007;.…”

Intravenous antagomiR‐494 lessens brain‐infiltrating neutrophils by increasing HDAC2‐mediated repression of multiple MMPs in experimental stroke

“…Given that our preceding bioinformatics analysis indicated that miR‐494 target genes are involved in the focal adhesion molecule pathway, 13 we investigated the expression of the classical adhesion molecules CD11b and MMP9 in the peripheral neutrophils of AIS patients and analyzed their relationship with the miR‐494 levels. Consistent with bioinformatics analysis, both CD11b and MMP9 were prominently increased in neutrophils of AIS patients and were negatively associated with miR‐494 expression in neutrophils (Figure 2A‐D; P < .05).…”

“…Venous blood samples from AIS patients and healthy controls were collected into K3EDTA tubes, and neutrophils were separated by a standard Ficoll‐Paque Plus gradient method as previously reported 13 . Then, neutrophils were suspended in 0.5 mL of TRIzol and frozen at −80°C prior to the test.…”

“…As a promising novel class of small regulatory molecules, microRNAs have been found to play pivotal roles in the inflammatory process associated with ischemic stroke, 10 and accumulating evidence has indicated the value of miRNAs in diagnosis, prognosis and treatment of ischemic stroke 11,12 . Recently, we found that miR‐494 levels in the blood was robustly upregulated in acute ischemic stroke (AIS) patients, and intravenous administration of antagomiR‐494 alleviated neuronal and sensorimotor functional damage in the mouse model of ischemic stroke, through reducing T helper cell type 1 (Th1) shift via targeting the histone deacetylase 2 (HDAC2)‐STAT4 pathway 13 . HDAC2 belongs to the class I HDACs and is known to act as epigenetic and transcriptional regulators.…”

“…HDAC2 belongs to the class I HDACs and is known to act as epigenetic and transcriptional regulators. It was already known that HDAC2 could regulate the phenotype shift of T helper cells, microglia, and macrophages 13‐15 . However, whether miR‐494 and its target HDAC2 are implicated in brain infiltration of neutrophil or phenotypic transformation following ischemic stroke is not clear.…”