The effect of human GRIN1 gene 5' functional region on gene expression regulation in vitro.

INTRODUCTION: Abnormal expression of ionotropic glutamate receptor NMDA type subunit 1, the key subunit of the NMDA receptor, may be related to many neuropsychiatric disorders. In this study, we explored the functional sequence of the 5' regulatory region of the human GRIN1 gene and discussed the transcription factors that may regulate gene expression. MATERIALS AND METHODS: Twelve recombinant pGL3 vectors with gradually truncated fragment lengths were constructed, transfected into HEK-293, U87, and SK-N-SH cell lines, and analyzed through the luciferase reporter gene assay. JASPAR database is used to predict transcription factors. RESULTS: In SK-N-SH and U87 cell lines, regions from -337 to -159 bp, -704 to -556 bp inhibited gene expression, while -556 to -337 bp upregulated gene expression. In HEK-293 and U87 cell lines, the expression of fragment -1703 to + 188 bp was significantly increased compared to adjacent fragments -1539 to + 188 bp and -1843 to + 188 bp. The protein expressions of fragments -2162 to + 188 bp and -2025 to + 188 bp, -1539 to + 188 bp and -1215 to + 188 bp, -1215 to + 188 bp and -1066 to + 188 bp were significantly different in HEK-293 and SK-N-SH cells. According to the predictions of the JASPAR database, the transcription factors REST, EGR1, and CREB1/HIC2 may bind the DNA sequences of GRIN1 gene from the -337 to -159, -556 to -337, and -704 to -556, respectively. In addition, zinc finger transcription factors may regulate the expression of other differentially expressed fragments. CONCLUSIONS: Abnormal transcription regulation in the proximal promoter region of GRIN1 (-704 to + 188 bp) may be involved in the course of neuropsychiatric diseases.

Functional Analysis of the 3' Untranslated Region of the Human GRIN1 Gene in Regulating Gene Expression in vitro.

PURPOSE: Abnormal expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor may potentially increase the susceptibility to neuropsychiatric diseases. The purpose of this study was to investigate the functional sequence of the 3'UTR of the human GRIN1 gene, which encodes the GluN1 receptor to determine the effect on the expression of GluN1 receptor. METHODS: We transferred seven recombinant pmirGLO recombinant vectors containing the 3'UTR truncated fragment of the GRIN1 gene into HEK-293, SK-N-SH, and U87 cell lines and compared the relative fluorescence intensity of adjacent length fragments. The TargetScan database was used to predict miRNAs. Then, miRNA mimics/inhibitors were co-transfected into the three cell lines with the 3'UTR of GRIN1 (pmirGLO - GRIN1), to investigate their influence on GRIN1 gene expression. RESULTS: Compared with the pmirGLo-Basic vector, the relative fluorescence intensity of the complete GRIN1 gene 3'UTR recombinant sequence -27 bp - +1284 bp (the next base of the stop codon is +1) was significantly decreased in all three cell lines. The relative fluorescence intensities were significantly different between -27 bp - +294 bp and -27 bp - +497 bp regions, and between -27 bp - +708 bp and -27 bp - +907 bp regions. According to the prediction of the TargetScan database and analysis, miR-212-5p, miR-324-3p and miR-326 may bind to +295 bp - +497 bp, while miR-491-5p may bind to +798 bp - +907 bp. After co-transfection of miRNA mimic/inhibitor or mimic/inhibitor NC with a recombinant vector in the 3'UTR region of GRIN1 gene, we found that has-miR-491-5p inhibited GRIN1 expression significantly in all three cell lines, while has-miR-326 inhibitor upregulated GRIN1 expression in HEK-293 and U87 cells. CONCLUSION: miR-491-5p may bind to the 3'UTR of the GRIN1 gene (+799 bp - +805 bp, the next base of the stop codon is +1) and down-regulate gene expression in HEK-293, SK-N-SH, and U87 cell lines, which implicates a potential role of miR-491-5p in central nervous system diseases.

Transcription Factor CEBPB Inhibits the Expression of the Human HTR1A by Binding to 5' Regulatory Region in Vitro.

This study identified a transcription factor that might bind to the 5' regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the -1219 to -1209 bp (ATG+1) region of the HTR1A. Two sequences located in the -722 to -372 bp and -119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence -1219 - -1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.

Rs1625579 polymorphism in the MIR137 gene is associated with the risk of schizophrenia: updated meta-analysis.

The Schizophrenia Psychiatric GWAS Consortium (PGC) has identified the rs1625579 polymorphism in the MIR137 gene, which encodes miR-137, as the strongest new association with schizophrenia in the European population. However, whether the influence of rs1625579 on schizophrenia in the Asian population is consistent with these results remains unclear. A total of 21 studies (9878 schizophrenic patients and 9447 control subjects) that met the inclusion criteria were included in our meta-analysis. Pooled analysis, subgroup analysis, sensitivity analysis and publication bias were performed. The T allele, TT genotype and TT + GG genotype were associated with schizophrenia as risk factors. Subgroup analysis shows that no heterogeneity existed in the European and Asian populations. Our meta-analysis found that the Rs1625579 polymorphism in the MIR137 gene was associated with the risk of schizophrenia. The current findings provide a reference for case-control studies of schizophrenia in the future.

Investigation of control region sequences of mtDNA in a Chinese Maonan population.

DNA sequences in the control region of mitochondrial DNA (mtDNA) were investigated in 206 unrelated individuals living in Huanjiang Maonan Autonomous County in the People's Republic of China. DNA was extracted from blood-stained filter papers. Hypervariable regions, including HVI and HVII, were amplified and sequenced and sequences aligned and compared with the revised Cambridge sequence (rCRS). One hundred and seventy-two polymorphic sites were identified that defined 170 haplotypes. Of these, 143 were unique and 27 were shared by more than one individual. Genetic diversity was estimated to be 0.9977 (± 0.0008), and the random match probability was 0.71%. The proportions of macro-haplogroups R*, M*, N*, D, U, R0, L3*, and L* were 50.49%, 26.21%, 11.17%, 3.88%, 3.88%, 2.43%, 1.46%, and 0.49%, respectively. Additionally, phylogenetic comparison and principal component analysis (PCA) demonstrated that Chinese Maonans shared a close genetic relationship with the Gelao ethnic community in Laos and China. These results may be useful in future human genetic studies and forensic examinations.