Mechanism of ARPP21 antagonistic intron miR-128 on neurological function repair after stroke

Abstract

Objective: Stroke is a cerebrovascular disorder that often causes neurological function defects. ARPP21 is a conserved host gene of miR-128 controlling neurodevelopmental functions. This study investigated the mechanism of ARPP21 antagonistic intron miR-128 on neurological function repair after stroke.

Methods: Expressions of ARPP21 and miR-128 in stroke patients were detected. The mouse neurons and astrocytes were cultured in vitro and treated with oxygen-glucose deprivation (OGD). The OGD-treated cells were transfected with pc-ARPP21 and miR-128 mimic. The proliferation of astrocytes, and the apoptosis of neurons and astrocytes were detected, and inflammatory factors of astrocytes were measured. The binding relationship between miR-128 and CREB1 was verified. The rat model of middle cerebral artery occlusion (MCAO) was established. ARPP21 expression in model rats was detected. The effects of pc-ARPP21 on neuron injury, brain edema volume, and cerebral infarct in rats were observed.

Results: ARPP21 expression was downregulated and miR-128 expression was upregulated in stroke patients. pc-ARPP21 facilitated the proliferation of astrocytes and inhibited apoptosis of neurons and astrocytes, and reduced inflammation of astrocytes. miR-128 mimic could reverse these effects of pc-ARPP21 on neurons and astrocytes. miR-128 targeted CREB1 and reduced BDNF secretion. In vitro experiments confirmed that ARPP21 expression was decreased in MCAO rats, and pc-ARPP21 promoted neurological function repair after stroke.

Conclusion: ARPP21 upregulated CREB1 and BDNF expressions by antagonizing miR-128, thus inhibiting neuronal apoptosis and promoting neurological function repair after stroke. This study may offer a novel target for the management of stroke.

FIGURE 1

ARPP21 was downregulated in patients with stroke. (A/B) Expressions of ARPP21 and miR‐128 in peripheral blood serum of stroke patients and healthy volunteers were detected using RT‐qPCR. The experiment was repeated three times independently. Data were expressed as mean ± standard deviation and analyzed using t test, *p < 0.05.

FIGURE 2

Astrocytes were incubated and identified in vitro. (A) Morphological changes in neurons and astrocytes were observed under the inverted phase‐contrast microscope. (B) Proliferation ability of astrocytes was detected using MTT assay. (C) Neurons and astrocytes were identified using immunofluorescence. (D) Expressions of ARPP21 and miR‐128 in cells were detected using RT‐qPCR. The cell experiment was repeated three times independently. Data were expressed as mean ± standard deviation. Data in panel (B/D) were analyzed using t test, **p < 0.01.

FIGURE 3

Overexpression of ARPP21 facilitated the proliferation of astrocytes, and inhibited apoptosis of neurons and astrocytes. pc‐ARPP21 and miR‐128 mimic were transfected into OGD‐treated neurons and astrocytes. (A) Expressions of ARPP21 and miR‐128 in cells were detected using RT‐qPCR. (B) Proliferation ability of astrocytes was detected using MTT assay. (C) GFAP and MAP2 expression was detected using Western blotting. (D) Cell apoptosis was measured using flow cytometry. The cell experiment was repeated three times independently. Data were expressed as mean ± standard deviation, and analyzed using one‐way ANOVA, followed by Tukey’s multiple comparisons test, **p < 0.01.

FIGURE 4

Overexpression of ARPP21 reduced cellular inflammation and alleviated oxidative stress. pc‐ARPP21 and miR‐128 mimic were transfected into OGD‐treated neurons and astrocytes. (A) Levels of TNF‐α, IL‐6, and IL‐1β in astrocytes were detected using ELISA. (B) Content of ROS was determined by fluorescence probe DCFH‐DA. (C–E) Contents of GSH, SOD, and MDA were detected using the kits. The cell experiment was repeated three times independently. Data were expressed as mean ± standard deviation, and analyzed using one‐way ANOVA, followed by Tukey’s multiple comparisons test, **p < 0.01.

FIGURE 5

miR‐128 targeted CREB1. (A) The binding site of miR‐128 and CREB1 was predicted by bioinformatics website. (B) The binding relationship between miR‐128 and CREB1 was verified using dual‐luciferase reporter gene assay. (C) Expression of miR‐128 was detected using RT‐qPCR. The cell experiment was repeated three times independently. Data were expressed as mean ± standard deviation and analyzed using one‐way ANOVA, followed by Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01.

FIGURE 6

ARPP21 was downregulated in the brain tissues of MCAO rats. (A) Expression and localization of ARPP21 in rats were determined using immunofluorescence. (B) Expressions of ARPP21, CREB1, and BDNF were detected using Western blotting. (C) Expression of miR‐128 was detected using RT‐qPCR. N = 18. Data were expressed as mean ± standard deviation, and analyzed using one‐way ANOVA, followed by Tukey’s multiple comparisons test, **p < 0.01.

FIGURE 7

Overexpression of ARPP21 reduced infarct size and alleviated brain injury in MCAO rats. pcDNA‐ARPP21 and pcDNA‐NC were transfected into MCAO rats. (A) expression of ARPP21 was detected using RT‐qPCR. (B) Neurologic score was determined by the Bederson’s scale. (C) Analysis of brain edema. (D/E) infarct size was evaluated using TCC and HE staining. (F) Expression of miR‐128 was detected using RT‐qPCR. (G) mRNA expressions of CREB1 and BDNF were detected using RT‐qPCR. N = 18. Data were expressed as mean ± standard deviation, and analyzed using one‐way ANOVA, followed by Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01.