IS often causes a strong neuroinflammatory response and oxidative stress response, which causes irreversible neuronal damage and disability in patients. Although blood vessels can be recanalized clinically, cerebral ischemia reperfusion itself usually causes neuronal damage to be difficult to recover22. Reducing inflammation and oxidative reactions provides new ideas for the prevention or treatment of cerebral ischemia-reperfusion injury23. However, there is still a lack of effective targets for down-regulating inflammation, oxidative stress and the resulting neurodegeneration. In our study, we mainly focused on the role of key miRNAs in patients with IS.
We first screened the changes in mRNA and miRNA expression profiles of patients with IS through the analysis of public databases. We found 111 DEmiRNA and 501 DEmRNA. The highly enriched TFs of DEmiRNAs include SP1, EGR1, SP4 and so on. The GO enrichment of DEmRNAs is mainly distributed in two categories: activation of neutrophil activity and activation of immune response. Further KEGG enrichment revealed several important pathways, including hematopoietic cell lineage, cell adhesion molecules, phagosomes, and T cell receptor signaling. Animal experiments have shown that stroke activates hematopoietic cells by increasing sympathetic tone24. Anti-adhesion agents also seem to expand the therapeutic window of thrombolytic therapy25. In systemic infections and traumatic brain injuries, transient disturbances in the phagocytic function of phagocytes and related bactericidal mechanisms have been detected26,27. The immune changes caused by stroke can impair the defense function of phagocytes against bacteria. These changes increase the chance of infection28, and T cells respond to the protective effects of acute inflammatory injury and acute stroke29. Animal experiments have shown that B-cell-deficient mice have a larger infarct area and more severe functional defects, suggesting that enhanced B-cell regulation may be a new way to treat this destructive neurological disease30. In conclusion, we believe that hematopoietic cell lineage, cell adhesion molecules, phagosomes, and T cell receptor signaling pathways may play an important role in the pathogenesis of IS.
MiR-34c-5p, miR-125a-5p, miR-455-5p and miR-361-5p have high expression levels in the construction of miRNA-mRNA regulatory network and ceRNA regulatory network. Further SVM model construction, screening miR-34c-5p, miR-140-3p, miR-330-3p can be used as disease prediction markers. At the same time, we recruited patients and healthy volunteers, collected peripheral blood, and tested the expression levels of key miRNAs. The results of the experiment were consistent with the previous bioinformatics analysis. In addition, we had developed a nomogram that can be used to predict the probability of illness based on the information that are easy to collect when visiting a doctor and the relative expression of important miRNAs. The internal verification in the queue showed good discrimination ability.
Hsa-miR-140-3p is a microRNA related to the regulation of inflammation, oxidative stress and apoptosis31,32. In recent reports, miR-140-3p has played a pathogenic or protective role in a variety of diseases, including myocardial infarction33, hyperglycemia34, skeletal dysplasia 35, equine metabolic syndrome 36, neonatal repetitive pain37, stent restenosis, and coronary artery disease38. Importantly, it was found that miR-140-3p is involved in the regulation or imbalance of oxidative stress and apoptosis in myocardial infarction39,40. This disease has many similarities with IS.
It has been reported that miR-34c-5p directly regulates the expression of soluble guanylate cyclase β under the induction of hypoxia. They found that the key TF Sp1 controls the expression of miR-34c-5p during hypoxia41. The latest research also proved that overexpression of miR-34c-5p can improve cerebral infarction in a rat model of middle cerebral artery occlusion, and miR-34c-5p plays an important role in cerebral ischemia/reperfusion injury 42. MiR-330-3p has become a therapeutic target and biomarker for neurological diseases43. Inhibition of miR-330-3p inhibits the progression of neuropathic pain by inhibiting neuro-inflammation in the body44. However, the further mechanism of anti-inflammatory of miR-140-3p, miR-34c-5p, miR-330-3p, anti-oxidative stress or anti-apoptotic effects should be further studied.
miR-140-3p, miR-34c-5p, miR-330-3p have a high degree in the network we constructed. Experiments verified that the expression of miR-140-3p, miR-34c-5p, and miR-330-3p is higher in the IS population. These miRNAs also play an important role in the pathogenesis and progression of IS. They may be potential new therapeutic targets for cerebral ischemic stroke.
Nowadays, nomograms are widely used in medicine field. The nomogram leverages a simple numerical interface, higher accuracy, and easier-to-calculate predictions to help make better clinical decisions45. Our research has developed and validated a new predictive tool that uses only four readily available variables and the relative expression levels of key miRNAs to assess the risk of IS in patients. In the analysis of risk factors, the level of cholesterol, the level of low-density lipoprotein, high blood pressure, diabetes, the expression status of miR-34c-5p, miR-140-3p, and miR-330-3p may be related to the disease status of patients with cerebral infarction. The nomogram shows that high cholesterol content, high low-density lipoprotein content, high blood pressure, diabetes, relatively high expression of miR-34c-5p, miR-140-3p, and miR-330-3p may be key individual factors in determining the risk of IS. This is similar to previous research46,47. Higher level of low-density lipoprotein is also associated with higher risk of disease. The study showed that patients with relatively high expression of key miRNAs, miR-34c-5p, miR-140-3p, and miR-330-3p are more likely to suffer from cerebral infarction.
We have developed an effective tool for predicting the risk of cerebral infarction. This tool can help clinicians identify high-risk patients early, and diagnose and treat patients early through personalized risk prediction and intervention. In addition, early intervention, such as controlling risk factors, improving diet and living habits, will benefit patients at high risk of disease at the beginning of treatment48.
Our current research also has some limitations. First, we only used qRT-PCR to verify the expression level of miRNA, and the study of its mechanism requires more experiments in future research. Second, although the robustness of our nomogram has been extensively checked by internal verification using guided procedure testing, external verification cannot be performed. Third, the generality of other cerebral infarction populations in other regions and countries is uncertain. It requires external evaluation in the wider population of cerebral infarction.