Identification of hsa-miR-1275 as a Novel Biomarker Targeting MECP2 for Human Epilepsy of Unknown Etiology

Abstract

Epilepsy affects around 70 million people worldwide, with a 65% rate of unknown etiology. This rate is known as epilepsy of unknown etiology (EUE). Dysregulation of microRNAs (miRNAs) is recognized to contribute to mental disorders, including epilepsy. However, miRNA dysregulation is poorly understood in EUE. Here, we conducted miRNA expression profiling of EUE by microarray technology and identified 57 pathogenic changed miRNAs with significance. The data and bioinformatic analysis results indicated that among these miRNAs, hsa-microRNA (miR)-1275 was highly associated with neurological disorders. Subsequently, new samples of serum and cerebrospinal fluid were collected for validation of hsa-miR-1275 expression by TaqMan assays. Results show that hsa-miR-1275 in serums of EUE were increased significantly, but in cerebrospinal fluid, the miRNA was decreased. Moreover, the MECP2 gene was selected as a hsa-miR-1275 target based on target prediction tools and gene ontology analysis. Validation of in vitro tests proved that MECP2 expression was specifically inhibited by hsa-miR-1275. Additionally, overexpression of hsa-miR-1275 can elevate expression of nuclear factor κB (NF-κB) and promote cell apoptosis. Taken together, hsa-miR-1275 might represent a novel biomarker targeting MECP2 for human EUE.

Keywords: MECP2; epilepsy of unknown etiology; hsa-miR-1275; hsa-miR-132.

Graphical abstract

Figure 1

Differentially Regulated miRNA in Epilepsy of Unknown Cause (EUE) Samples (A) Principal-component analysis (PCA) of all samples of the dataset. mRNA-based PCA clearly separates EUE patients (S1 and S2) (shown in red) from the normal control sample C1 (shown in blue). (B) Cluster dendrogram of control serum sample (C1) and EUE patients’ serum samples (S1 and S2). (C) Venn diagram shows the overlap between the two EUE patients’ serum samples.

Figure 2

hsa-miR-1275 Was Frequently Differentially Regulated in EUE (A) Analysis of cellular components of hsa-miR-1275 by ClueGO following a Benjamini-Hochberg correction. (B) Detection of hsa-miR-1275 in serum of EUE by TaqMan assays. (C) Detection of hsa-miR-1275 in cerebrospinal fluid by TaqMan assays. hsa-miR-132 was a positive control, and cel-miR-39 was used as the housekeeping gene for the detection of hsa-miR-1275 expression.

Figure 3

Effect of Exogenous hsa-miR-1275 on MECP2 Expression In Vitro (A) Gene and pathway interaction networks. The green nodes are the hub genes; the blue triangles are signal pathways. The red circles were for the corresponding edges to highlight the crosstalk between hsa-miR-1275 and MECP2. All pathways represented here are highly significant following a Benjamini-Hochberg correction. Pathway enrichment analysis and visualization were performed in Cytoscape version 3.0.2. (B) Binding sites of 3′ untranslated region (3′ UTR) with MECP2 and sequence alignment between multiple species. (C) The complementary relationship of the seed sequence of hsa-miR-1275 and 3′ UTR of MECP2 (425–431). (D) qRT-PCR detection of the transcription of MECP2 after transfection with hsa-miR-1275 mimic, mimic negative control (mimic NTC), and cotransfection with the inhibitor of hsa-miR-1275 and inhibitor NTC. (E) Representative western blot results of MECP2 and semiquantifications of the western blot bands by ImageJ software. (F) Luciferase reporter assays to validate the targeting 3′ UTR of MECP2 by hsa-miR-1275. The 3′ UTR of MECP2 (and mutant) with the hsa-miR-1275 binding region was cloned and inserted into the pmirGLO vector. The U251 cells were cotransfected with hsa-miR-1275 mimics or mimic NTCs. Cells were analyzed for luciferase activity after 48 h. For each transfection, luciferase activity was averaged from three replicates. Data were expressed as mean ± SD. ∗p < 0.05.

Figure 4

Overexpression of hsa-miR-1275 Promotes Cell Apoptosis (A) DAPI attaining nucleus of U251 transfected with (top) hsa-miR-1275 mimic NTC (100 nM) and (bottom) mimic (100 nM). Cells: original magnification, 100×. (B) Detection of cell apoptosis by flow cytometry (Annexin V FITC-propidium iodide [PI]). (C) The transcription level of NF-κB affected by hsa-miR-1275. U251 cells were cotransfected with pNFκB-TA-luc and hsa-miR-1275 mimic (50 nM), inhibitor (100 nM), or NTCs. For each transfection, luciferase activity was averaged from three replicates. Data were expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

Figure 5

Involvement of the hsa-miR-1275-MECP2 Interaction in Human EUE Network of the predicted hsa-miR-1275-MECP2 signal in human EUE. MECP2 was supposed to be a potential target for hsa-miR-1275 in EUE regulation. MECP2 plays a critical role in neuronal maturation, synaptic plasticity, axons and dendrites, and neuron growth by alteration of brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF1), serum glucocorticoid-inducible kinase 1 (SGK), FK506-binding protein 5 (FKBP5), cytotoxic necrotizing factor 1 (CNF1), and mechanistic target of rapamycin (mTOR), etc. By binding to the 3′ UTRs of MECP2, hsa-miR-1275 might inhibit MECP2 expression. Simultaneously, hsa-miR-1275 positively regulates NF-κB expression and promotes cell apoptosis.