MiR-340-5p alleviates neuroinflammation and neuronal injury via suppressing STING in subarachnoid hemorrhage

Abstract

Background: Subarachnoid hemorrhage (SAH) is a severe acute neurological disorder. SAH causes neuroinflammation and leads to early brain injury (EBI) and secondary injury. MicroRNAs are crucial regulators in a variety of neurological diseases. This study was performed to decipher how miR-340-5p functions in SAH.

Methods: An experimental mouse model with SAH was established by the intravascular perforation, and the in vitro SAH model was constructed by exposing cocultured primary neurons and microglia to oxyhemoglobin. After overexpression of miR-340-5p in mice, the neurobehavioral disorders were evaluated by Garcia test; brain edema was evaluated by wet-dry method; blood-brain barrier (BBB) damage was detected with Evan's blue staining; levels of inflammatory cytokines were detected with enzyme-linked immunosorbent assay. After miR-340-5p was transfected in to microglia, Iba-1 expression was detected by Western blot, and neuronal apoptosis were detected with flow cytometry. The targeting relationship between miR-340-5p and STING was verified by dual-luciferase reporter gene assay and RNA immunoprecipitation assay.

Results: MiR-340-5p was significantly inhibited in the brain tissues of mice with SAH and microglia of SAH model, and neurological impairment, brain edema, BBB injury, and neuroinflammation were significantly alleviated in mice after overexpressing miR-340-5p. STING was identified as a target of miR-340-5p, and STING overexpression could counteract the effects of miR-340-5p overexpression on neurons.

Conclusion: MiR-340-5p can attenuate EBI caused by SAH-induced neuroinflammation by inhibiting STING.

Keywords: STING; early brain injury; miR-340-5p; neuroinflammation; subarachnoid hemorrhage.

FIGURE 1

Experimental design of the present work

FIGURE 2

SAH causes neurological injury, neuroinflammation, and miR‐340‐5p inhibition in mice. (a) The neurobehavioral scores of mice in sham group and SAH group were evaluated by Garcia test method. (b) The brain water content of sham SAH mice was measured by dry and wet method. (c) Evan's blue staining was used to detect BBB injury of the mice. (d) The contents of IL‐1 β, IL‐6, and TNF‐α in the brain tissue of mice were determined by ELISA. (e) The expression levels of IL‐1β, IL‐6, and TNF‐α mRNA in the brain tissue of mice were assessed by qRT‐PCR. (f) The expression of miR‐340‐5p in the brain of the mice was evaluated by qRT‐PCR. *p < .01 and p < .001

FIGURE 3

miR‐340‐5p overexpression alleviates SAH‐induced nerve injury and neuroinflammation in mice. (a) After intracerebroventricular injection of miR‐340‐5p mimic, miR‐340‐5p expressions in the sham group, SAH group, SAH + miR‐control group, and SAH + miR‐340‐5p mimic group were measured by qRT‐PCR. (b) The neurobehavioral scores of mice in sham group, SAH group, SAH + miR‐control group, and SAH+miR‐340‐5p mimic group were evaluated by Garcia test. (c) The brain water content of the mice in sham group, SAH group, SAH + miR‐control group, and SAH + miR‐340‐5p mimic group was determined by dry–wet method. (d) Evan's blue was used to detect BBB injury of the mice in sham, SAH, SAH + miR‐control, and SAH+miR‐340‐5p mimic groups. (e) The expression of IL‐1β, IL‐6, and TNF‐α in the brain of mice in sham group, SAH group, SAH + miR‐control group, and SAH+miR‐340‐5p mimic group was evaluated by qRT‐PCR. (f) The levels of IL‐1β, IL‐6, and TNF‐α in midbrain tissue of mice in sham group, SAH group, SAH + miR‐control group, and SAH+miR‐340‐5p mimic group were measured by ELISA. *p < .01 and p < .001

FIGURE 4

Overexpression of miR‐340‐5p alleviates OxyHb‐induced neuroinflammation and neuronal apoptosis. (a) The expression of miR‐340‐5p in microglia of control group, OxyHb group, OxyHb + miR‐control group, and OxyHb + miR‐340‐5p mimic group was determined by qRT‐PCR. (b) The contents of IL‐1β, IL‐6, and TNF‐α in microglia of control group, OxyHb group, OxyHb + miR‐control group, and OxyHb + miR‐340‐5p mimic group were detected by qRT‐PCR. (C) IL‐1β, IL‐6, and TNF‐α levels of microglia in the control, OxyHb, OxyHb + miR‐control, and OxyHb + miR‐340‐5p mimic groups were determined by ELISA. (d) LDH assay was used to assess the cytotoxicity of neurons in the control, OxyHb, OxyHb + miR‐control, and OxyHb + miR‐340‐5p mimic groups. (e) The expression of Iba‐1 in microglia of control group, OxyHb group, OxyHb + miR‐control group, and OxyHb + miR‐340‐5p mimic group was determined by Western blot. (f) The expression of caspase‐3 in neurons of control group, OxyHb group, OxyHb + miR‐control group, and OxyHb + miR‐340‐5p mimic group was determined by qRT‐PCR to assess neuronal apoptosis. (g) Flow cytometry was used to determine the rate of apoptotic neurons in the control, OxyHb, OxyHb + miR‐control, and OxyHb + miR‐340‐5p mimic groups. *p < .001

FIGURE 5

miR‐340‐5p binds directly to STING. (a) TargetScan predicted that the sequence of miR‐340‐5p matched the sequences of STING 3′ UTR. The WT‐STING and MUT‐STING luciferase reporter vectors were constructed. (b) MiR‐340‐5p mimic or miR‐control and WT‐STING or MUT‐STING were cotransfected into 293T cells. Forty‐eight hours later, luciferase activity was measured to validate the predicted binding site. (c) RIP assay was performed to verify the direct interaction between STING 3′UTR sequence and miR‐340‐5p. (d) The expression of STING in the microglia of control, OxyHb, OxyHb + miR‐control, and OxyHb + miR‐340‐5p mimic groups were detected by Western blot. *p < .001

FIGURE 6

Upregulation of STING can weaken the protective effect of overexpression of miR‐340‐5p. The neurons were divided into 5 groups: Control group, OxyHb group, OxyHb + miR‐control group, OxyHb + miR‐340‐5p mimic group, and OxyHb + miR‐340‐5p mimic + CMA group. (a) Western blot was used to detect the expression of STING in microglia in control group and CMA group. (b) qRT‐PCR was used to determine the expression levels of IL‐1β, IL‐6, and TNF‐α in microglia of each group. (c) The levels of IL‐1β, IL‐6, and TNF‐α in the supernatant of microglia in each group were determined by ELISA. (d) The expression of Iba‐1 in microglia of each group was detected by Western blot. (e) LDH assay was used to determine the injury of neurons in each group. (f) The expression of caspase‐3 in neurons of each group was determined by qRT‐PCR. (g) The level of neuronal apoptosis in each group was measured by flow cytometry. H. Phosphorylation levels of TBK1 in microglia of each group were determined by Western blot. *p < .01, and p < .001