Stress types include physical stress versus psychological stress as well as acute severe stress versus chronic mild stress [31, 32, 48, 55]. After undergoing psychological stress, stressors can be divided into stress susceptibility and stress resilience according to the different effects of stressors' physical and mental health. Susceptible mice exhibit symptoms such as fear memory, anxiety, and depression, whereas resilient mice fail to show these changes [39, 40]. Identifying the molecular mechanisms that lead some individuals to become susceptible to stress and others resilient is essential for developing effective treatments and, ultimately, preventing the emergence of fear of memory and anxiety [56]. In the present study, we focus on analyzing mRNA and microRNA profiles in the amygdala from the mice treated by psychological stress.
Susceptible mice compared to Resilient mice, the upregulated genes related to synapse elements in the amygdala include Oprm1, Vwf, Col6a3, Sv2c and Trhr, encode structural proteins of building up Peptide, Thyrotropin-releasing hormone and ECM receptors. The downregulated genes related to synapse elements in the amygdala include Scn7a, encodes structural proteins of building up Adrenergic synapse. The upregulated genes related to signaling pathways of regulating synapses in the amygdala include Pla2g4e, encodes structural proteins of building up Serotonergic and Glutamatergic synapses. The downregulated genes related to signaling pathways of regulating synapses in the amygdala include Cyp2c55, encodes structural proteins of building up Serotonergic synapse. In terms of the signaling pathways that regulate synapse functions, the downregulated genes in the amygdala include Grk1, Rac3 and Cd86, encode Chemokine, VEGF, Fc epsilon RI, Ras, MAPK, Wnt, Sphingolipid, B cell receptor, cAMP, Rap1, Toll-like receptor and Axon guidance signaling pathways. On the other hand, the upregulated genes include Ccr1, Ccr6, Oprm1, Trhr, Ccl9, Pla2g4e, Vwf, Col6a3, Robo3 and Eno1b, encode Chemokine, VEGF, Fc epsilon RI, Ras, MAPK, PI3K-Akt, Oxytocin, GnRH, HIF-1, Estrogen, Calcium and Axon guidance signaling pathways. Also in Cell adhesion molecules, the downregulated genes include Cd6 and Cd86, the upregulated genes include LOC547349 and H2-T10, encode Immune system. In Inflammatory mediator regulation of TRP channels, the downregulated genes include Cyp2c55, the upregulated genes include Pla2g4e, encode Inflammatory mediator. In Adherens junction, the downregulated genes include Rac3. In Cytokine-cytokine receptor interaction, the upregulated genes include Ccr1, Ccr6, LOC100861978 and Ccl9. In Long-term depression, the upregulated genes include Pla2g4e. In Cytosolic DNA-sensing pathway, the downregulated genes include Trex1. The data indicate that the upregulation of Peptide, Thyrotropin-releasing hormone and ECM receptor, Glutamatergic synapse, Cytokine-cytokine receptor interaction, Long-term depression, PI3K-Akt, Oxytocin, GnRH, HIF-1, Estrogen, Calcium signaling pathways in the amygdala may be related to psychological stress-induced susceptibility, and the downregulation may be related to resilience. The downregulation of Adrenergic synapse, Adherens junction, Wnt, Sphingolipid, B cell receptor, cAMP, Rap1, Toll-like receptor signaling pathways in the amygdala may be related to psychological stress-induced susceptibility, and the upregulation may be related to resilience.
In addition to analyzing the downregulation or upregulation of gene expression in specific signaling pathways, the imbalanced expression of genes in signaling pathways may be related to susceptibility and resilience. The data show that except for the expression of all genes in some signal pathways are downregulated or upregulated, genes in other signal pathways are partially upregulated and partially downregulated. For example, Serotonergic synapse, Cell adhesion molecules, Inflammatory mediator regulation of TRP channels, Chemokine, VEGF, Fc epsilon RI, Ras, MAPK, Axon guidance signaling pathways, this imbalanced expression may play a role in susceptibility caused by psychological stress. In the amygdala brain region, the expression of some genes in some signaling pathways is upregulated and the expression of other genes is downregulated in psychologically stressed mice, indicating that the molecular regulatory network in these pathways is imbalanced. In other words, the upregulation and downregulation of gene expression between multiple signaling pathways and in a single signaling pathway imbalances the molecular regulatory network within the amygdala, resulting in neuronal dysfunction in the amygdala. In summary, the upregulated genes of encoding synapse elements may strengthen the formation and transmission of synapses for susceptibility. The imbalance of signaling pathways may be involved in susceptibility.
In terms of the validation of our study and result, after the high throughput sequencing of mRNAs and microRNAs, We verified some mRNAs and microRNAs with qRT-PCR and analyzed the interaction between mRNAs and microRNAs. Our results indicate that the changed expression of mRNAs matches the changed expression of microRNAs well in high-throughput sequencing(Tables 4 ~ 9). Some genes with their altered expressions in high throughput sequencing have been confirmed by qRT-PCR analysis (Fig. 6 ~ 8 and 12). Furthermore, dual-luciferase report analysis has confirmed direct interactions between mRNA Tgoln2 and miRNA-24p-3p as well as between Pla2g4e and miRNA-24p-3p (Fig. 13). In a word, we are confident in our results, because is better than previous analyses in either microRNAs or mRNAs.
To the best of our knowledge, no study has yet reported an association between Tgoln2(Pla2g4e) and susceptibility. The research report shows, Tgoln2 encodes a transmembrane protein primarily localized to the trans-Golgi network (TGN), an important protein sorting station in the cell [57]. Tgoln2 is of particular interest because it was identified in a microarray expression study as having a consistently up-regulated expression pattern in the brains of suicide completers as compared to controls [58]. Pla2g4e belongs to cytosolic phospholipase A2 (cPLA2), which selectively catalyzes the hydrolysis of the sn-2 site of the glycerol portion of the membrane phospholipid to break the ester bond [59]. It is considered to be related to neural structure and function, a neurobehavioral disorder in research [60]. Some literature suggests that this gene may be a risk gene for panic disorder [61], there is also some literature indicating that there is a certain relationship between the occurrence of depression [62]. Cytosolic phospholipase A2 is closely related to axonal proliferation and excitotoxic damage under pathological conditions [63]. Various pathological changes in CNS disease lead to increased expression of cPLA2 and increased activity [63]. cPLA2 is involved in oxidative stress and inflammatory response in a variety of neurodegenerative diseases [62]. In normal brain cells, PLA2 regulation achieves a balance between the conversion of arachidonic acid to a pro-inflammatory mediator and reintegration into the membrane. Without strictly regulating PLA2 activity, a disproportionate pro-inflammatory mediator is produced. The resulting induced oxidative stress and neuroinflammation are similar to neurological diseases such as Alzheimer's disease, epilepsy, etc. [59, 63].
We found the changes of certain genes have not been observed in previous analyses[64–67], including Oprm1, Vwf, Col6a3, Sv2c, Trhr, Scn7a, Pla2g4e, Cyp2c55, Grk1, Rac3, Cd86, Ccr1, Ccr6, Ccl9, Robo3, Eno1b, Cd6, LOC547349, H2-T10, LOC100861978 and Trex1. The changes in the expression of these genes in the amygdala may be due to susceptibility and resilience.