The Mechanism of Long Non-coding RNA MEG3 for Neurons Apoptosis Caused by Hypoxia: Mediated by miR-181b-12/15-LOX Signaling Pathway

Abstract

Objective: lncRNAs are recently thought to play a significant role in cellular homeostasis during pathological process of diseases by competing inhibiting miRNA function. The aim of present study was to assess the function of long non-coding RNA (lncRNA) MEG3 and its functional interaction with microRNA-181b in cerebral ischemic infarct of mice and hypoxia-induced neurons apoptosis.

Methods: To address this question, we performed the experiments with in vivo middle cerebral artery occlusion (MCAO) mice model and in vitro oxygen-glucose deprivation (OGD)-cultured neuronal HT22 cell line. Relative expression of MEG3, miR-181b, and 12/15-LOX (lipoxygenase) mRNA was determined using quantitative RT-PCR. Western blot was used to evaluate 12/15-LOX protein expression. TUNEL assay was performed to assess cell apoptosis.

Results: In both MCAO mice and OGD-cultured HT22 cell, ischemia, or hypoxia treatment results in a time-dependent increase in MEG3 and 12/15-LOX expression and decrease in miR-181b expression. Knockdown of MEG3 contributes to attenuation of hypoxia-induced apoptosis of HT22 cell. Also, expression level of MEG3 negatively correlated with miR-181b expression and positively correlated with 12/15-LOX expression. In contrary to MEG3, miR-181b overexpression attenuated hypoxia-induced HT22 cell apoptosis, as well as suppressed hypoxia-induced increase in 12/15-LOX expression. By luciferase reporter assay, we concluded that miR-181b directly binds to 12/15-LOX 3'-UTR, thereby negatively regulates 12/15-LOX expression.

Conclusion: Our data suggested that long non-coding RNA MEG3 functions as a competing endogenous RNA for miR-181b to regulate 12/15-LOX expression in middle cerebral artery occlusion-induced ischemic infarct of brain nerve cells.

Keywords: 12/15-LOX; MCAO; MEG3; infarct; miR-181b.

Figure 1

Time course of genes expression in infarction area of middle cerebral artery occlusion mice and in hypoxia-stimulated HT22 neural cell line. After a series of 6, 12, and 24 h animal establishment, quantitative RT-PCR was performed to determine the relative expression of (A) MEG3, (B) miR-181b, and (C) 12/15-LOX mRNA; (C) representative blots of 12/15-LOX protein expression by Western blot. HT22 cells were experienced 6, 12, and 24 h of glucose deprivation for following gene expressional analysis. Relative expression of (D) MEG, (B) miR-181b, and (E) 12/15-LOX, and (F) 12/15-LOX protein expression were determined. Data are presented as mean ± sd. ^*P < 0.05, ^**P < 0.01 compared with sham with no MCAO surgery or cell with no treatment.

Figure 2

Manipulation of MEG3 affects hypoxia-induced apoptosis in HT22 cell. HT22 cells were pre-transfected with si-MEG3 or si-NC for 24 h and then deprived 12 h of glucose-free DMEM. (A) Cell apoptosis was evaluated by TUNEL assay. Expression level of (B) miR-181b and (C) 12/15-LOX mRNA and protein were determined. Also cells were transfected with pcDNA-MEG3 or pcDNA empty plasmid for 24 h. (D) Cell apoptosis, expression level of (E) miR-181b and (F) 12/15-LOX mRNA and protein were evaluated. Data are presented as mean ± sd. ^**P < 0.01 compared to cell with no hypoxia treatment or pcDNA; ^##P < 0.01 compared with hypoxia +si-NC.

Figure 3

Functional interaction of MEG3 and miR-181b mediates HT22 cell apoptosis. HT22 cells were pre-treated with miR-181b mimic or pre-NC before exposed to glucose deprivation. (A) Cell apoptosis and (B) expression level of 12/15-LOX mRNA and protein were determined. (C) Cell apoptosis and (D) 12/15-LOX expression were analyzed in miR-181b inhibitor treated HT22 cells. Cells were then co-treated with pcDNA-MEG3 and miR-181b mimic. (E) Expression level of 12/15-LOX and (F) cell apoptosis were determined. Data are presented as mean ± sd. ^**P < 0.01 compared to cell with no hypoxia treatment or NC or pcDNA; ^##P < 0.01 compared with hypoxia +pre-NC or pcDNA-MEG3+pre-NC.

Figure 4

Manipulation of cellular miR-181b regulates12/15-LOX expression in HT22 cell. (A) Representative base-pairs between 12/15-LOX 3′-UTR-WT and miR-181b with 12/15-LOX 3′-UTR-MU as positive control. (B) Cells were co-treated with 12/15-LOX 3′-UTR-WT or 12/15-LOX 3′-UTR-WT and miR-181b mimic; fluorescence activity and 12/15-LOX expression were determined. (C) Fluorescence activity and 12/15-LOX expression were determined in 12/15-LOX 3′-UTR-WT or 12/15-LOX 3′-UTR-WT and miR-181b mimic co-transfected HT22 cells. Data are presented as mean ± sd. ^**P < 0.01 compared with 2/15-LOX 3′-UTR-WT or NC.

Figure 5

si-MEG3 injection improves cerebral infarction. Mice were injected with lipid nanoparticles-formulated si-control or si- MEG3 for 24 h before MCAO establishment. (A) Relative expression of MEG3 in infract position was determined. (B) Infarct volume and edama volume and (C) motion function were evaluated. Examination of (D) relative expression of miR-181b and (E) 12/15-LOX in infract site. Data are presented as mean ± sd. ^**P < 0.01 compared with siNC.