MicroRNA-137 Inhibits EFNB2 Expression Affected by a Genetic Variant and Is Expressed Aberrantly in Peripheral Blood of Schizophrenia Patients

Abstract

MicroRNAs (miRNAs) are a class of endogenous and non-coding single-stranded RNAs of approximately 22 nucleotides, many of which are evolutionarily conserved. Genome-wide association studies have identified a robust statistical association between the MIR137 gene and schizophrenia in Europeans, which was replicated in the Han Chinese population in a case-control study. In the previous study, we provided evidence for a significant association between the EFNB2 gene and schizophrenia in Han Chinese subjects. In the current study, we utilized computational analysis, vector construction of point mutations, luciferase reporter assays and gene expression assays including RT-qPCR and western blotting methods to investigate miR-137 directly targeting EFNB2 gene and explore the reversal effect of a genetic variant of SNP rs550067317 in the putative seed-pair region of EFNB2 3'-UTR. We also found that miR-137 could be detected in the peripheral blood of a cohort of first-onset schizophrenia patients and healthy controls, and the area under curve was 0.795 (95% confidence interval 0.700-0.890), which is a middle diagnostic value for disease, suggesting that it might be valuable for diagnosing schizophrenia. In summary, this study would improve our understanding of the role of miR-137 in schizophrenia-associated signaling pathways and identify the genetic basis of rs550067317 for schizophrenia. Furthermore, we provided new evidence for the involvement of miR-137 in the etiology and diagnosis of schizophrenia, which might contribute to the discovery of new biomarkers and therapeutic targets for the disease.

Keywords: EFNB2; MiR-137; Regulation; SNP; Schizophrenia.

Fig. 1

EFNB2 as target of miR-137, and different allele of SNP rs550067317 affects the MFE. A. Schematic diagram of the pairing relationship between miR-137 and EFNB2 mRNA 3′-UTR analyzed by TargetScan and Miranda online software. The putative seed-pair region was in red. B. Broad conservation of the target sites in EFNB2 3′-UTR among vertebrates. C. The location of SNP rs550067317 in EFNB2 3′-UTR binding site. D. Location of the SNP rs550067317 alleles.

Fig. 2

The results of restriction enzyme digestion and sequencing. A. The results of restriction enzyme digestion. Lane 1 shows the marker of D2000, lanes 2–4 shows the restriction enzyme digestion result of wild type/rs550067317(A), full mutant, rs550067317(C) construct, respectively. B, C, D. The sequencing result of wild type/rs550067317(A), full mutant, rs550067317(C) construct, respectively.

Fig. 3

MiR-137 directly targets EFNB2 gene and SNP rs550067317(C) independently reverses the inhibition. A. Schematic diagram of three different constructs. The arrow marked the minor allele C in the construct. B, C. Dual luciferase reporter assay of miR-137 mimics/negative co-transfected with full mutant and wild type construct in HEK293T cells after 24 h and 48 h. D. Dual luciferase reporter assay of miR-137 mimics/negative co-transfected with full mutant, rs55006731(C), and wild type construct in HEK293T cells. Bars represent SEM from three experiments. NC, negative control. ** indicates P < 0.01, *** indicates P < 0.001.

Fig. 4

The dual luciferase reporter assay of miR-137 mimics/negative co-transfecting with PmirGLO + ZNF804A in HEK293T cells. 24 h after transfection, the cells were assayed for firefly luciferase activity and Renilla luciferase activity test and the ratio of firefly/Renilla is obtained. Bars represent SEM from three experiments. NC, negative control. ** indicates P < 0.01.

Fig. 5

MiR-137 inhibits endogenous expression of EFNB2 at protein level. A. RT-qPCR results of miR-137 relative expression in SH-SY5Y cells. B. RT-qPCR results of EFNB2 mRNA relative expression SH-SY5Y cells. C. Western blotting results of EFNB2 and GAPDH as an internal control. The band size of EFNB2 is 50 kDa which is consistent with the specification of the antibody kit. D. Statistical analysis of Western blotting results from three independent western blotting experiments. NC, negative control. * indicates P < 0.05, ***indicates P < 0.001.

Fig. 6

MiR-137 is aberrantly expressed in peripheral blood of schizophrenia patients. A. The distribution of sample ages. B. The expression of miR-137 in the peripheral blood of schizophrenia patients and healthy controls. C. The expression of EFNB2 in the peripheral blood of schizophrenia patients and normal controls. D. Receiver operating characteristic (ROC) curve analysis of the diagnostic value of miR-137 expression for schizophrenia. CON, normal control; SCH, schizophrenia patients; AUC, area under curve. ***indicates P < 0.001.