miR-339 Promotes Hypoxia-Induced Neuronal Apoptosis and Impairs Cell Viability by Targeting FGF9/CACNG2 and Mediating MAPK Pathway in Ischemic Stroke

Abstract

Ischemic stroke (IS) is a common cerebrovascular disease characterized by insufficient blood blow to the brain and the second leading cause of death as well as disability worldwide. Recent literatures have indicated that abnormal expression of miR-339 is closely related to IS. In this study, we attempted to assess the biological function of miR-339 and its underlying mechanism in IS. By accessing the GEO repository, the expression of miR-339, FGF9, and CACNG2 in middle cerebral artery occlusion (MCAO) and non-MCAO was evaluated. PC12 cells after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment were prepared to mimic in vitro the IS model. The levels of miR-339, FGF9, CACNG2, and MAPK-related markers were quantitatively measured by qRT-PCR and Western blot. CCK-8 and flow cytometry analyses were performed to examine cell viability and apoptosis, respectively. IS-related potential pathways were identified using KEGG enrichment analysis and GO annotations. Bioinformatics analysis and dual-luciferase reporter assay were used to predict and verify the possible target of miR-339. Our results showed that miR-339 expression was significantly increased in MCAO and OGD/R-treated PC12 cells. Overexpression of miR-339 inhibited cell viability of PC12 cells subjected to OGD/R treatment. FGF9 and CACMG2 are direct targets of miR-339 and can reverse the aggressive effect of miR-339 on the proliferation and apoptosis of OGD/R-treated PC12 cells. Moreover, miR-339 mediated the activation of the MAPK pathway, which was inhibited by the FGF9/CACNG2 axis in PC12 cells treated by OGD/R stimulation. In summary, these findings suggested that miR-339 might act as a disruptive molecule to accelerate the IS progression via targeting the FGF9/CACNG2 axis and mediating the MAPK pathway.

Keywords: FGF9/CACNG2; MAPK pathway; apoptosis; ischemic stroke; miR-339; oxygen-glucose deprivation/reoxygenation (OGD/R).

Figure 1

miR-339 plays an aggressive role on IS progression. (A) Analysis of miR-339 expression in MCAO patients (n = 3) and normal samples (n = 3) on the basis of GEO database. Data were presented as the mean (SD), P < 0.05 vs. control group. (B) Measurement of miR-339 expression in PC12 cells after OGD/R treatment using qRT-PCR. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. normal group. (C) CCK-8 assay was performed to detect the proliferation of OGD/R-induced PC12 cells with different transfections, Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. normal group, ^##P < 0.01 vs. OGD/R group.

Figure 2

FGF9 and CACNG2 might serve as the potential targets of miR-339 in IS. (A) KEGG enrichment analysis and (B) GO annotations were conducted to identify the pathways related with down-regulated genes of IS. (C) Venn curve of the interaction between the putative targets that predicted by TargetScan and down-regulated genes. (D) FGF9 and (E) CACNG2 expressions were determined owing to the GEO datasets (accession number: GSE61616, includes five normal controls and five MCAO cases), P = 0.0079.

Figure 3

miR-339 can negatively regulate the expression levels of FGF9 and CACNG2. (A) The predicted binding site between miR-339 and FGF9. The luciferase activity assay was conducted to examine the luciferase activity of WT FGF9 and MUT FGF9 in PC12 cells transfected with miR-339 mimic or miR-339 inhibitor. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. negative control (NC) group. (B) Sequence of interaction site between miR-339 and CACNG2. Dual-luciferase reporter assay was implemented to assess the relative luciferase activity of WT CACNG2 and MUT CACNG2 in PC12 cells after miR-339 mimic or miR-339 inhibitor transfection. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. NC group. (C,D) Detection of FGF9 mRNA and protein levels in PC12 cells with various transfections, including blank control, miR-339 mimic/inhibitor, pcDNA3.1-FGF9/si-FGF9, mimic+FGF9/inhibitor+si-FGF9. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. control group, ^##P < 0.01 vs. mimic+FGF9/inhibitor+si-FGF9. (E,F) Exploration of CACNG2 mRNA and protein levels in PC12 cells with different transfections, including blank control, miR-339 mimic/inhibitor, pcDNA3.1-CACNG2/si- CACNG2, mimic+CACNG2/inhibitor+si-CACNG2. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. control group, ^##P < 0.01 vs. mimic+CACNG2/inhibitor+si-CACNG2.

Figure 4

miR-339 aggravated OGD/R-induced injury through targeting FGF9 in PC12 cells. (A,B) CCK-8 assays were performed to detect the PC12 cell viability after OGD/R treatment and different transfections. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. OGD/R group, ^##P < 0.01 vs. OGD/R+mimic+FGF9 or OGD/R+inhibitor+si-FGF9 group. (C,D) Annexin V-fluorescein isothiocynate (FITC)/propidium iodide (PI) staining and flow cytometry analysis were implemented to evaluate cell apoptosis. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. OGD/R group, ^##P < 0.01 vs. OGD/R+mimic+FGF9 or OGD/R+inhibitor+si-FGF9 group.

Figure 5

The regulation of miR-339 on OGD/R-induced PC12 cell injury was modulated by CACNG2. (A,B) After transfection with various agents, the proliferative ability of PC12 cells stimulated by OGD/R was determined. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. OGD/R group, ^##P < 0.01 vs. OGD/R+mimic+CACNG2 or OGD/R+inhibitor+si-CACNG2 group. (C,D) The apoptotic potential of OGD/R-treated PC12 cells was measured using flow cytometry analysis. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. OGD/R group, ^##P < 0.01 vs. OGD/R+mimic+CACNG2 or OGD/R+inhibitor+si-CACNG2 group.

Figure 6

Effects of miR-339 on the MAPK pathway were associated with FGF9/CACNG2 in OGD/R-stimulated PC12 cells. (A–D) The immunoblots of p-P38 MAPK, P-38 MAPK, p-KNK, and JNK (left panels), and quantification of the gray values of corresponding protein bands (right panels) in PC12 cells after OGD/R treatment. Data were presented as the mean (SEM), n = 3, ^**P < 0.01 vs. OGD/R group, ^##P < 0.01 vs. OGD/R+mimic+FGF9/CACNG2 or OGD/R+inhibitor+si-FGF9/si-CACNG2 group.