Wu Xue, Ding Mei, Liu Yi, Xia Xi, Xu Feng-Ling, Yao Jun, Wang Bao-Jie
School of Forensic Medicine, China Medical University, Shenyang, China.
Front Mol Neurosci. 2019 Jan 31;12:13. doi: 10.3389/fnmol.2019.00013. eCollection 2019.
Abnormal expression of the 5-HT1A receptor, which is encoded by the HTR1A gene, leads to susceptibilities to neuropsychiatric disorders such as depression, anxiety, and schizophrenia. miRNAs regulate gene expression by recognizing the 3'-UTR region of mRNA. This study evaluated the miRNAs that might identify and subsequently determine the regulatory mechanism of HTR1A gene. Using the HEK-293, U87, SK-N-SH and SH-SY5Y cell lines, we determined the functional sequence of the 3'-UTR region of the HTR1A gene and predicted miRNA binding. Dual luciferase reporter assay and Western Blot were used to confirm the effect of miRNA mimics and inhibitors on endogenous 5-HT1A receptors. In all cell lines, gene expression of the -17 bp to +443 bp fragment containing the complete sequence of the 3'-UTR region was significantly decreased, although mRNA quantification was not different. The +375 bp to +443 bp sequence, which exhibited the most significant change in relative chemiluminescence intensity, was recognized by hsa-miR-3177-5p and hsa-miR-3178. In HEK-293 and U87 cells, hsa-miR-3177-5p significantly inhibited the 5-HT1A receptor expression, while a hsa-miR-3178 inhibitor up-regulated HTR1A gene expression in SK-N-SH and SH-SY5Y cells. By constructing the pmirGLO-vector with the mutated HTR1A gene, we further confirmed that hsa-miR-3177-5p recognized the HTR1A gene tgtacaca at +377 bp to +384 bp, and the +392 bp to +399 bp fragment cgcgccca was identified by hsa-miR-3178. hsa-miR-3177-5p and hsa-miR-3178 had significant inhibitory effects on expression of the HTR1A gene and 5-HT1A receptor and may directly participate in the development of neuropsychiatric diseases.
由HTR1A基因编码的5-HT1A受体异常表达会导致个体易患神经精神疾病，如抑郁症、焦虑症和精神分裂症。微小RNA（miRNA）通过识别信使核糖核酸（mRNA）的3'-非翻译区（3'-UTR）来调控基因表达。本研究评估了可能识别并随后确定HTR1A基因调控机制的miRNA。利用人胚肾293细胞（HEK-293）、U87细胞、人神经母细胞瘤细胞（SK-N-SH）和人神经母细胞瘤细胞（SH-SY5Y）系，我们确定了HTR1A基因3'-UTR区的功能序列并预测了miRNA结合情况。采用双荧光素酶报告基因检测和蛋白质免疫印迹法来证实miRNA模拟物和抑制剂对内源性5-HT1A受体的影响。在所有细胞系中，包含3'-UTR区完整序列的-17 bp至+443 bp片段的基因表达显著降低，尽管mRNA定量并无差异。hsa-miR-3177-5p和hsa-miR-3178识别出相对化学发光强度变化最为显著的+375 bp至+443 bp序列。在HEK-293和U87细胞中，hsa-miR-3177-5p显著抑制5-HT1A受体表达，而在SK-N-SH和SH-SY5Y细胞中，hsa-miR-3178抑制剂上调了HTR1A基因表达。通过构建携带突变HTR1A基因的pmirGLO载体，我们进一步证实hsa-miR-3177-5p识别HTR1A基因位于+377 bp至+384 bp处的tgtacaca，hsa-miR-3178识别+392 bp至+399 bp片段cgcgccca。hsa-miR-3177-5p和hsa-miR-3178对HTR1A基因和5-HT1A受体的表达具有显著抑制作用，可能直接参与神经精神疾病的发生发展。
