Abstract
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.
Highlights
When the endogenous Dicer enzyme was knocked down, the inhibitory function of the −17 bp to +443 bp sequence was apparently disappeared in SK-N-SH and U87 cell lines (Figure 1)
In the SK-N-SH and SH-SY5Y cell lines, the hsa-miR-3178 inhibitor significantly increased expression of the endogenous 5-HT1A receptor, which was significantly different compared with the miR-NC inhibitor (p = 0.037 and 0.015, respectively; Figures 6, 7). This experiment revealed an important functional sequence located at +375 bp to +443 bp in the 3 -UTR region of the HTR1A gene
We found that the down-regulation of the HTR1A gene 3 -UTR sequence was firmly restored in the SK-N-SH and U87 cell lines that knocked down the Dicer enzyme
Summary
The serotonin nervous system is associated with the pathological mechanisms in various neuropsychiatric disorders, such as depression (Lemonde et al, 2003; Dorado et al, 2007), anxiety (Heisler et al, 1998; David et al, 2005) and schizophrenia (Gu et al, 2013; Lin et al, 2015; Takekita et al, 2016). 5-HT neurons originate in the raphe nuclei of the brain and regulate emotions and mood by dominating 5-HT1A receptor-rich regions, including the cortex, hippocampus, diaphragm, and amygdala (Törk, 1990; Ou et al, 2000; Lemonde et al, 2004). Related animal and autopsy studies have confirmed that a reduction in 5-HT1A postsynaptic receptors and hsa-miR-3177-5p and hsa-miR-3178 Inhibit 5-HT1A over-expression of 5-HT1A presynaptic receptors (autoreceptors), which decrease the activity and content of serotonin neurotransmitters at an overall level, are present in patients with major depression (Arango et al, 2001; Miller et al, 2009; Parsey et al, 2010). Evidence has suggested that the number of 5-HT1A auto-receptors in depressive suicidal patients was reduced by 43% (Arango et al, 2001). Autopsy studies have determined that there is an increase in 5-HT1A receptors in the prefrontal cortex of patients with schizophrenia (Burnet et al, 1996; Sumiyoshi et al, 1996). Irregular expression of the 5-HT1A receptor may be an important factor causing susceptibility to developing neurological diseases
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call