Effective biomarkers and therapeutic targets of nerve-immunity interaction in the treatment of depression: an integrated investigation of the miRNA-mRNA regulatory networks

Introduction

Major depressive disorder (MDD) is a crippling mental illness characterized by depression and emotional disturbance brought on by a variety of psychological factors [1]. The high prevalence, recurrence, and disability rates are all difficult issues to address in MDD prevention and treatment [2]. Depression currently affects at least 20% of the world’s population, and the WHO predicts that depression will overtake heart disease as the leading disorder by 2030 [3]. The occurrence of MDD not only reduces patients’ quality of life, but also has a significant financial impact on the families and society. Hypotheses based on monoamine transmitter, immune-inflammation, the stress, neuroplasticity are now being researched [4]. In recent years, scholars have placed a greater emphasis on the study of the pathophysiology of immunity-inflammation and MDD [5]. According to various studies, the activation of the peripheral immune system is associated with MDD [6]. However, the pathobiology of MDD inflammation is poorly understood, and additional research is needed to identify the accurate diagnostic biomarkers. Chronic moderate stress, neuroinflammation, and immune response changes have all been connected to the genesis of MDD [7]. More and more studies have shown that the neurological system is inextricably linked to the immune system.

Chemical medications are currently used primarily in the clinical treatment of MMD in Western medicine, whereas Chinese materia medica, acupuncture and moxibustion are commonly used in traditional Chinese medicine (TCM). Traditional antidepressants, on the other hand, can only relieve a portion of MMD patients’ symptoms, and 20% to 30% of MMD patients do not respond to antidepressant medications. Neuroinflammation is thought to be one of the causes for ineffective treatment [8]. Following an injury, neuroinflammation occurs as a result of an imbalance between the synthesis and release of pro-inflammatory and anti-inflammatory cytokines from central or peripheral sources [9]. The most visible aspect of neuroinflammation is the activation of microglia. Microglia activation results in the synthesis of nutritional and anti-inflammatory chemicals under physiological conditions. Microglia shows excessive activation in pathological condition such as chronic stress or infection, resulting increased not only in levels of inflammatory chemicals in the brain, but also neuronal damage and death [10]. In order to effectively manage neuroinflammation and restore neurotropism and neurotransmitter function, it is critical for basic and clinical MMD research to identify specific diagnostic biomarkers from the perspective of nerve-immunity interaction.

Endogenous intracellular and extracellular small non-coding RNAs are known as microRNAs (MiRNAs). It may govern cell proliferation, division, apoptosis, and metabolism as regulators of numerous biological processes by promoting the degradation of mRNAs and inhibiting their translation, so that its targeted mRNAs play a negative feedback regulatory function [11]. Liguori discovered that combining microRNA and mRNA expression analysis in pediatric multiple sclerosis is an integrated approach to uncovering novel disease pathogenic mechanisms [12]. Many other studies have shown a close relationship between microRNAs and mRNAs in disease regulation, particularly the negative feedback regulation of targeted mRNAs by their miRNAs [13–14], which allows us to further investigate the mechanisms and functions of miRNAs in relation to MMD development. MiRNAs have been investigated as potential therapeutic targets for a variety of central nervous system diseases due to their involvement in nearly all fundamental cellular activities. Changes in miRNA expression are well known to be linked to the pathogenesis of numerous neurodegenerative diseases and hold significant therapeutic promise in the treatment of mood disorders, including clinical MDD [15]. Detecting the differences in pathogenic miRNA levels in the brain will be an excellent method for identifying people with early MMD. Bioinformatics is a critical tool for furthering biological understanding and therapeutic development [16].

In MMD, it has been reported that miRNAs are not only involved in the pathogenesis of the disease [17], but are also associated with depression caused by various illnesses [18, 19], as well as the response to specific treatments such as isoliquiritin [20], oligonucleotides [21], or purinergic receptor [22], confirming their potential as disease monitoring biomarkers. So far, little miRNAs research has been done in the Nerve-immunity Interaction of Depression. Therefore, understanding the role of miRNA in the development of MMD may lead to the discovery of a biomarker that can be used as a neuroimmune interaction in the treatment of MDD. The work is performed on microarray data and Figure 1 show Framework.

Materials and Methods

Results

The purpose of this study is to investigate, evaluate, and summarize the evidence of nerve-immunity-related miRNA in the plasma of MMD patients, so as to determine useful biomarkers, new therapeutic targets, and prognosis evaluation methodologies in the nerve-immunity interaction therapy of MDD. We predicted miRNAs and constructed a miRNA-mRNA network.

Discussion

Due to the stresses of work and life, the prevalence of MMD is growing year by year. Patients with MMD not only suffer from the disorder, but also have an enormous burden on their families and society as a whole. According to researches, nerve-immunity network dysfunction generates excessive production of inflammatory components and stimulates glucocorticoid release, both of which contribute to the initiation of MMD [23]. Increased expression of pro-inflammatory factors such as TNF-α, IL-6, and IL-1 in MMD patients’ and animal models’ serum, cerebrospinal fluid, and hippocampus forms a negative feedback regulation of the anti-inflammatory factor IL-10, resulting in neuronal inflammation and neuronal damage in the central nervous system [24].

At the moment, academics place a high value on nerve-immunity interaction treatment MDD. Nerve-immunity interaction therapy is associated with commonly used therapeutic therapies, such as escitalopram, fluoxetine, acupuncture, and Chinese materia medica. Common antidepressants, such as escitalopram and fluoxetine, have been shown to improve central function by increasing central serotonergic nerve activity. It can also successfully regulate the levels of brain-derived neurotrophic factors and inflammatory factors in patients’ blood [25]. Acupuncture can increase the content of serum 5-HT, up-regulate the content of anti-inflammatory cytokine IL-10, and reduce the content of proinflammatory cytokine IL-6 and TNF-α [26]. Chinese materia medica such as Rhizoma Cyperi, Rhizoma Coptidis, Cinnamomum, and Radix Paeoniae Alba can improve neuronal growth and development by mediating neurotrophic factors and other signal pathways, resist MMD-induced neuronal damage, and regulate signal transduction pathways such as NLR, ErbB, and chemokines, thus regulating inflammatory and immune responses, resulting in antidepressant effects [27]. Although it has been demonstrated that miRNAs play an important role in the initiation and progression of MMD, the link between miRNAs and mRNA remains unknown. As a result, it is critical to investigate MMD at the molecular level and to discover the mechanism of nerve-immunity interaction therapy of MDD, thus providing reference ideas for future fundamental research and clinical application.

We discovered 5 hub DE-mRNAs: KCND2, MYT1L, GJA1, CHL1, and SNAP25, which were all up-regulated genes. Both hsa-miR-206 and hsa-miR-338-3p, which are down-regulated in MMD, are similar miRNAs. Although 5 pairs of regulatory relationships between miRNA and mRNA have never been reported before, this study validated hsa-miR-206 with GJA1, SNAP25, hsa-miR-338-3p with KCND2, MYT1L via the miRDB database, and hsa-miR-338-3p via the TargetScan database, and predicted the possibility of interaction between 5 pairs of miRNA-mRNA. It provided theoretical groundwork for further experimental confirmation of the interaction of these miRNA and mRNA.

MiRNAs are tiny non-coding RNAs that directly regulate more than 30% of the genes in cells by inhibiting the degradation or translation of target mRNAs [28]. It links to nearly every fundamental biological function, including MMD and anxiety disorders [29]. Changes in miRNA expression are thought to be connected to the pathogenesis of a variety of neurodegenerative diseases [30]. MiR-206, a member of the muscle-specific miR-1 family, was first thought to regulate skeletal muscle embryonic development [31]. MiR-338-3p was first thought to be a cancer-related miRNA, but it was later found to be involved in neuronal regulation, including the Parkinson’s disease pathway [32]. From the neurological aspect: BDNF participates in neuroplasticity and protection throughout brain development, as well as playing an important role in emotion-related brain activities [33]. MiR-206 can suppress the production of BDNF following transcription, which is vital for the regulation and participation in MMD [34]. It was also discovered that miR-206 antagonists could attenuate stress-induced aggressive behavior while increasing BDNF expression in SI mice. Up-regulation of miR-206 has been demonstrated to impair neuron function and result in poor adaptive behavior [35]. MiR-338 can promote myelination in the nervous system, and over-expression of miR-338 can enhance oligodendrocyte differentiation while inhibiting myelin negative regulators Sox6 and Hes5, therefore promoting myelin formation [36]. MiR-338-5p has been found to safeguard the cognitive function of transgenic APP/PS1 mice by lowering neuronal death [37]. Other researches have shown that silencing miR-338-5p can result in neuronal polarity loss and a considerable decrease in the number of neurons. Thus, activating miR-338, to some extent, can influence the emotional function of the brain [38]. From the immune aspect: MiR-206 also links to the TNF signaling pathway [39]. The activated TNF pathway mediated by miR-206 can, in turn, drive NF-κB to enter the nucleus and boost the creation and release of inflammatory mediators such as TNF-α, IL-8, IL-6, and others to involve in inflammatory and immunological processes [40]. TNF inflammatory cytokines, which are plentiful in tumor microenvironments, can promote tumor growth, disrupt cell proliferation and death, and impair the innate immune response to cancer cells [41]. MiR-338-3p links to the TNF signaling pathway as well. TNF may induce inflammation by stimulating the NF-κB/MAPK signaling pathway and decreasing miR-338-3p levels [42]. Over-expression of miR-338-3p has been shown to block the ERK/p38MAPK signaling pathway and reduce the expression of pro-inflammatory genes VCAM-1 and ICAM-1RNA [43].

KCND2 is a potassium voltage-gated channel subfamily D protein-coding gene that can encode a type A potassium channel protein, which is important during the repolarization stage of an action potential [44]. By controlling potassium transport across the excitatory membrane in the brain, voltage-gated potassium channels regulate dendritic A-type current I (SA) in brain neurons [45]. Myt1L is a member of the Myt/NZF family, which encodes a neuronal transcription factor with 6 zinc fingers. Its expression has only been found in brain tissue thus far [46]. This gene is important to neural differentiation, and mutations in it have been linked to autism spectrum disorder and an autosomal dominant form of cognitive dysfunction [47]. Mutations in Myt1L in chromosomal band 2p25.3 have been linked to intellectual disability, while Myt1L repetition has been linked to schizophrenia and MMD [48]. Gap junction alpha-1 protein (GJA-1) expression had proven to be down-regulated in MDD patients, as was the Cx43 mRNA that encoded It [49]. CHL1 is a cell adhesion molecule from the L1 family. The deletion and repetition of this gene have been linked to a number of neurological disorders, including autism, Parkinson’s disease, and mental disability [50]. This study revealed that the level of serum CHL1 in patients with MMD declined dramatically and was inversely proportional to the severity of the disease, implying that CHL1 may be involved in the pathogenesis of MMD and immune cell dysfunction [51]. SNAP-25 has been proved to be associated with the intensity of depressive symptoms in patients with MMD as judged by the Beck MMD Rating Scale in numerous studies [52]. Both the hub miRNA and the mRNA created by constructing a miRNA-mRNA network may have a role in the incidence and progression of MMD via nerve-immunity interaction.

We explored the major targets of MDD using GO and KEGG enrichment to study the molecular mechanism. According to GO and KEGG enrichment analyses, the key targets contributed value in immunological and neuronal aspects of BP, CC, and MF. 83 CC mainly involves the presynaptic, synaptic membrane, neuronal cell body, synaptic connections between neurons, and voltage-gated sodium channel complex. 84 MF mainly involves immune receptor function, neurotransmitter receptor activity and chemokine binding. 470 BP mainly involves neutrophil, T cell and B cell activation, activation of surface receptors and immune response signal pathways, and regulation of chemical synaptic transmission. In addition, 39 MMD-related pathways, including immunological and neuroregulatory pathways, were identified and assessed.

Among the signaling pathways studied in this study, the signal pathways mentioned below are linked to immune and neurological regulation. The B cell and T cell receptor signal pathways, the chemokine signal pathway, and the JAK-STAT signal pathway. Th1 and Th2 cell differentiation, and Th17 cell differentiation are all involved. The human immune system’s Th17, Th1, and Th2 CD4+T lymphocyte subsets have critical roles in promoting inflammation and immunosuppression, respectively, and contribute to immune system homeostasis while supporting B cell activation and playing a role in humoral immunity. ① Th17 cell differentiation: Th17 contributes to the pathogenesis of MDD by boosting autoantibody production and the predisposition for autoimmunity [53]. Furthermore, Th17 and Treg are connected in differentiation and function, and they are frequently in a state of dynamic equilibrium. If the proportion of them is no longer balanced, it will result in aberrant immunological responses such as inflammatory, tumor and autoimmune reactions [54]. Th17 had been proven to be elevated in the brains of MMD model mice, but RORt defective mice demonstrated resistance to learning helplessness. As a result, an increase in Th17 can cause MMD, whereas suppressing Th17 production or function can lessen the risk of MMD in mice [55]. ② Th1 and Th2 cell differentiation: Both Th1 and Th2 cells can release cytokines, which can boost their proliferation while inhibiting each other to maintain a relative equilibrium. When the equilibrium is disrupted, also known as "Th1/Th2 drift," it can be expected to result in the emergence and development of a variety of disorders associated with immunological escape [56]. ③ T and B signaling pathways: In MMD patients, the immunological function was compromised, for T cell apoptosis increased, neutrophils rose, and total lymphocytes decreased. T cell function degradation could be due to inflammatory substances such as tumor necrosis factor-α destroying T cell function. B cells mediate humoral immunity via antibodies; as the number of B cells falls, so does the rate of transformation [57]. ④ Chemokine signaling pathway: the major purpose of chemokines is to control leukocyte migration (homing) to their proper regions during inflammation and homeostasis. Some control immune cell chemotaxis during the immune surveillance process, while others attract neutrophils to the site of infection or tissue injury for anti-infection, which acts as a directing role in the innate and adaptive immune systems. Furthermore, chemokines have an important role in the progression of disease [58]. ⑤ JAK/STAT signal pathway: This is not only one of the most important transmembrane signal transduction routes, but also the most important signal pathway activated by inflammatory cytokines [59]. Furthermore, JAK and STAT are key components of many signaling pathways that regulate cell growth, differentiation, survival, and pathogen resistance, including those involving the IL-6 (gp130) receptor family, which aids in the regulation of B cell differentiation, plasma cell production, and acute phase response [60]. Neural aspect: The JAK/STAT signaling pathway is widely implicated in the proliferation and differentiation of neural stem cells (NSC) throughout central nervous system development, and it is important in controlling hippocampal nerve reorganization [61]. A member of its family, the JAK2/STAT3 signal pathway, is involved in neuronal death and nervous reorganization in ischemic encephalopathy [62]. Numerous studies have revealed that TNF-α activates JAK/STAT signal pathway via the protein-gp130 on the cell membrane’s surface, and so contributes to the control of central nervous system degenerative alterations [63]. In addition, the JAK/STAT signaling pathway can influence neuron reorganization via inflammatory cytokines, BDNF, neurotransmitters, etc., [64]. As a consequence, medications and other treatment interventions can suppress neuronal death and promote nerve reorganization by altering the JAK/STAT signal pathway’s associated molecules.

Existing knowledge holds that the central nervous system is usually regarded as an immune zone due to the blood-brain barrier. However, as more researches are conducted, this idea is increasingly changing [65]. Chemokines, which include Th17, Th1, and Th2, are key chemicals in the immunological and neurological systems. They can coordinate this migration of T and B cells to inflammatory areas, promote T cell activity and help B cell activation, and play a role in regulating immunity-inflammation in the immune system [66]. Following psychological stress, activated T cells can enter the central nervous system in a variety of ways, reduce inflammation, and support neuronal integrity via cellular immunity, thus modifying depressed behavior [67]. Furthermore, the JAK/STAT signaling system influences brain remodeling by activating inflammatory cytokines and altering the metabolism of the neuro-endocrine-immune network.

Depending on the integrated analysis of miRNA-mRNA regulatory networks, miR-338-3p and miR-206 can coordinate the regulation of the neuro-endocrine-immune network by regulating chemokines such as Th17, Th1 and Th2, immune cells such as T/B and the JAK/STAT signaling pathway, among other things. The realization of bi-directional regulation of the HPA axis, which can normalize nerve conduction, minimize immuno-inflammatory responses, and restore normal neural, endocrine, and immunological function, plays a therapeutic role in MMD. To research useful biomarkers and treatment targets from the perspective of MMD and the host neuro-endocrine-immune network to regulate the level of immune-inflammatory cytokines in the microenvironment and balance neuro-endocrine-immune homeostasis at the molecular level. It is crucial to improve the critical mechanism of neuro-endocrine-immune imbalance in MMD patients.

There are quite a few papers published in recent years focusing on the relationship between miRNA and MDD [68–69]. However, compared to previous studies, we have focused more on the neurological and immunological aspects. First of all, we have made a new addition to miRNA regarding MMD in neuroimmunity compared to previous articles. Secondly, we used a joint analysis of multiple GEO data, although there were individual differences, we performed a batch Normalize data correction before the statistics to improve the stability and confidence of the results. It also reduced the small sample bias caused by the reduced sample size. Third, we not only performed TF analysis (including BP, CC, (MF)) using FunRich, but also GO and KEGG analyses, which allowed the results to be verified against each other to increase the feasibility of the results. Our research has limitations, although it provides theoretical underpinnings and research suggestions. First, we were unable to acquire sufficient external data from other publicly available sources to evaluate model’s dependability. Second, we focused more on miRNAs linked with neuroimmunity in our early expression analyses, but did not undertake any functional or mechanistic research. What’s more, although we batched Normalize various GEO datasets before analysis, it has little effect on the results, and there may still be bias when compared to analysis from the same dataset. We will undertake further research at a later time. Finally, no MDD studies were conducted to validate the relationship between prognostic genes and miRNA. As a result, additional research is required to confirm the above-mentioned findings and limitations.

Conclusions

To summarize, MMD occurs and progresses as a result of multi-target, multi-pathway, multi-signal pathway, and multi-mechanism interactions, and the regulatory process is synergistic and bi-directional. MiRNA affects the production of KCND2, Myt1L, GJA-1, CHL1, and SNAP-25, which can activate or inhibit the B cell receptor signal pathway, T cell receptor signal pathway, and chemokine signal pathway. JAK-STAT and other signaling pathways control immune-inflammatory responses, neuron remodeling, and other processes, as well as affecting the onset and course of MMD.

Despite the fact that this study provides some theoretical underpinnings and research suggestions for nerve-immunity interaction, it has several limitations. The following suggestions are given for future improvement: ① Because the current data is derived from the GEO database, it is challenging to determine the dependability and quality of the statistical data. In the future, the number of data sources will be increased while decreasing the data offset. ② Conduct more scientific and clinical research to determine whether TCM therapies such as acupuncture and other therapies can enhance MMD patients’ neurological and immunological function by regulating the amount of miRNA in the brain.

Supplementary Materials

Supplementary Tables 1-4 and 10

Supplementary Tables 5-9 and 11