title: Experimental validation of candidate schizophrenia gene CALN1 as a target for microRNA-137.
abstract: MIR137, which encodes microRNA-137 (miR-137), and several of its target genes exhibit genome-wide significant associations with schizophrenia. In a previous study, we analyzed the SNPs in a group of predicted MIR137 target genes and detected genome-wide significant association of schizophrenia with rs2944829 in the CALN1 gene. However, no experimental evidence for CALN1 and MIR137 interaction has yet been reported. In this study, we first computationally analyzed the putative miR-137 target site on CALN1 and predicted that miR-137 binds CALN1 at nucleotide (nt) position 236-242 in the 3'UTR. Then we assayed gene expression by transfecting miR-137 mimics into HEK293 and SH-SY5Y cell lines. Quantitative real-time RT-PCR results showed that the expression level of CALN1 significantly decreased in cells co-transfected with miR-137 mimics compared to cells transfected with the blank control (P=.0046 in HEK293 cell lines, P=.038 in SH-SY5Y cells lines). Finally, we co-transfected different combinations of miRNA mimics and either wild type CALN1 3'UTR or mutant 3'UTR reporters into HEK293 and SH-SY5Y cell lines and assessed the specificity of miRNA binding using a luciferase reporter assay. The transfection of miR-137 mimics corresponded with a considerable reduction of luciferase activity on vectors carrying the target fragment (P=1.17×10(-5), 68% reduction in HEK293 cell line, and P=5.09×10(-6), 32% reduction in SH-SY5Y cell line). This inhibition was impaired by site-directed mutagenesis of the miR-137 target fragment. Our results provide strong evidence that CALN1 is a target of miR-137.
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Shihui
authors.initials: S
authors.lastname: Xia
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Xinyao
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Teng
authors.initials: T
authors.lastname: Wang
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Qiaoli
authors.initials: Q
authors.lastname: Li
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Qinghe
authors.initials: Q
authors.lastname: Xing
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China.
authors.forename: Lin
authors.initials: L
authors.lastname: He
authors.affiliation: Children's Hospital and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China. Electronic address: xzzhao@fudan.edu.cn.
authors.forename: Xinzhi
authors.initials: X
authors.lastname: Zhao
mesh_terms: D020413:3' Untranslated Regions; D002147:Calmodulin / Q000235:genetics*; D045744:Cell Line, Tumor; D019295:Computational Biology; D015536:Down-Regulation; D017930:Genes, Reporter; D056726:Genetic Association Studies; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D012559:Schizophrenia / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; C428830:CALN1 protein, human; D002147:Calmodulin; C545567:MIRN137 microRNA, human; D035683:MicroRNAs; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
keywords: GWAS; Luciferase; Real-time PCR; Schizophrenia; miRNA