In recent years, the view that miRNAs participate in the pathophysiological process of mental diseases by regulating hundreds of target transcripts has been widely accepted. Evidence showed that the dysregulation of many miRNAs in postmortem brain samples was associated with schizophrenia (Moreau et al. 2011). However, it is difficult to exclude the effects of long-term treatment and medication for the explanation of the altered miRNA expression in the postmortem brain or peripheral circulation(Luoni and Riva 2016; Santarelli et al. 2013; Seo et al. 2014; Wang et al. 2019). In an article studying the association between miR-195 level and cognitive impairment in patients with schizophrenia of different genders, Huang et al. also demonstrated that recruiting first-episode and drug-free patients as the object could rule out the effects of antipsychotics on cognition and miRNA expression levels (Huang et al. 2020).
Therefore, this study recruited FES patients who were diagnosed for the first time and had not been treated with antipsychotic drugs, analyzed the alteration of miRNA expression profiles in their peripheral blood, predicted target mRNAs and integrated their common functions. We initially screened the differentially expressed miRNAs in schizophrenia patients through high-throughput sequencing globally, subsequently performed a qRT-PCR procedure on 4 miRNAs (miR-9-5p, miR-328, miR-144, and miR-4467) in an expanded sample for verification to avoid false-positive results. We found that compared with healthy controls, the level of miR-9-5p in peripheral blood of FES patients was down-regulated, and miR-4467 was up-regulated. Moreover, the ROC curve indicated that, as a non-invasive biomarker of FES, the expression level of miR-9-5p in peripheral blood showed a better diagnostic value than miR-4467.
There have been reported cases earlier in researches focusing on the two miRNAs and mental diseases. miR-4467 was first found in the cerebrospinal fluid of Alzheimer's patients and was identified as a potential biomarker for Alzheimer (Denk et al. 2015). Disorders of the brain can exhibit similar symptoms(Anttila et al. 2018), which made us wonder if the etiology of schizophrenia and Alzheimer would overlap. It is worth mentioning that this study is the first attempt that the alteration of miR-4467 expression was explored in SCZ. As for miR-9-5p, Mehmet et al. detected a significant up-regulation of miR-9-5p in blood samples of patients with schizophrenia using qRT-PCR(Camkurt et al. 2016). Studies have also shown that miR-9-5p is an important regulator of neurogenesis(Krichevsky et al. 2006; Leucht et al. 2008; Shibata et al. 2011), nerve cell proliferation and differentiation (Delaloy et al. 2010; Sun et al. 2013; Zhao et al. 2009), and axon development (Dajas-Bailador et al. 2012; Otaegi et al. 2011). A genomic analysis published in JAMA Psychiatry revealed that among several miRNAs that regulate schizophrenia risk genes, the miR-9-5p target is the most enriched, provided evidence for its role in the etiology of schizophrenia (Hauberg et al. 2016).
In order to better understand the role of miR-9-5p and miR-4467 in schizophrenia, we performed bioinformatics analysis to predict their target genes. In addition to the top 10 functions of potential target genes shown in Fig. 3, the SCZ-related biological processes that miR-9-5p may participate in also include regulation of astrocyte, oligodendrocyte differentiation, regulation of neural precursor cell and mesenchymal cell proliferation, regulation of neuron differentiation, development, and apoptotic, the biosynthetic process of ceramide and sphingomyelin, neuron-neuron synaptic transmission, regulation of dendritic spine development, the neuronal stem cell population maintenance, brain development, motor learning, branching morphogenesis of a nerve, axonogenesis (all p < 0.05). And predicted cell components also include neuron projection, dendritic spine synapse, synaptic vesicle membrane, and postsynaptic membrane. Enrichment of the KEGG pathway showed that the target genes of miR-9-5p were enriched in the neurotrophin signaling pathway and estrogen signaling pathway. It is worth mentioning that, A number of recent studies have demonstrated that SCZ is a neurodevelopmental disorder (Kranz et al. 2015; Li et al. 2020; Su et al. 2021). And Estrogen is believed to play a vital role in the pathophysiology of neurodevelopmental disorders (Crider and Pillai 2017), including involvement in brain development, synaptic plasticity and neuroprotection, while neurons are the primary site of estrogen synthesis in the brain (Azcoitia et al. 2011; Cui et al. 2013). Therefore, the potential of the pathways as targets for the treatment of SCZ can be further explored. Since there were only 92 target genes of miR-4467 predicted in this study, no statistically significant pathway enrichment results were produced. Nevertheless,the BP of miR-4467 target gene was obviously enriched in regulation of neuron differentiation and development, and negative regulation of Schwann cells proliferation.
We further screened the hub genes of miR-9-5p and miR-4467 by the Maximal Clique Centrality algorithm (MCC). The hub genes can be classified according to their functions, resulting in psychosis susceptibility genes (NEDD4, EIF4G1, FBXL16)(Deng et al. 2015; Han et al. 2019; Smith et al. 2019), neurodevelopment-related genes (EIF4G1, FBXL3) (Ansar et al. 2019; Dong et al. 2020), mental disease phenotype-related genes (CPLX1) (Glynn et al. 2007; Glynn et al. 2005), neuroprotective genes (FEM1A) (Fujikawa et al. 2016), and antipsychotic drug-sensitive gene (BCL2L1) (Fatemi et al. 2012).
Taken together, the enrichment analysis and hub genes indicated that miR-9-5p and miR-4467 are likely to play a role in neurodevelopment and synaptic transmission via the regulation of target genes, which could contribute to the occurrence and development of schizophrenia. In addition, considering the involvement of target gene groups in the regulation of neurodevelopment, it further proves the significance of selecting first-episode schizophrenia patients to explore the etiology and pathogenesis of schizophrenia.
For miR-328-3p and miR-144-3p whose expression changes were also detected in the sequencing samples, studies have been verified in autistic spectrum disorder patients (Kichukova et al. 2017; Nt et al. 2018) and Alzheimer’s disease (Zhou et al. 2019). However, there is no evidence to support the abnormal expression of miR-328-3p or miR-144-3p in FES patients, and no significant changes in the expression of the two were found in our validation set, the trends were different from the sequencing results (p > 0.05). In this regard, we considered that high-throughput sequencing and qRT-PCR are two different experimental platforms. High-throughput sequencing is used for large-scale screening, reflecting the overall gene expression trend of the samples, and there is a certain probability of false positives. There is no guarantee that the trend of the gene expression is exactly the same as that of qRT-PCR.
In spite of our novel findings of alterations of miR-4467 and miR-9-5p in first-episode schizophrenia patients. This study has some limitations that need to be acknowledged. First of all, our case-control study design has determined the fact that it cannot reflect the causal relationship between miRNA expression level and schizophrenia. Secondly, although we have enrolled enough samples based on the pre-experiment results and statistical power requirements, larger sample will definitely help us provide more valuable data, we are still recruiting. Of further interest are the expression level of miR-9-5p and miR-4467 predicted targets in the peripheral blood of FES patients, as well as the binding sites of miRNA and target genes. Are these hub genes directly regulated by miR-9-5p and miR-4467? What are their binding fragments? These problems need to be explored through subsequent investigations at the cellular level.
In conclusion, miR-9-5p and miR-4467 in peripheral blood are found to be potential biomarkers for early diagnosis of schizophrenia, and might affect the onset and development of SCZ by target regulation of neurodevelopment-related mRNAs.