Analysis of the expression level and predictive value of CLEC16A|miR-654-5p|RARA regulatory axis in the peripheral blood of patients with ischemic stroke based on biosignature analysis

 Why the fuck are you doing predictions rather than the work that will get survivors recovered?

Laziness? Incompetence? Or just don't care? NO leadership? NO strategy? Not my job? Not my Problem?

Analysis of the expression level and predictive value of CLEC16A|miR-654-5p|RARA regulatory axis in the peripheral blood of patients with ischemic stroke based on biosignature analysis

Jiang-jie Hao¹Yuan Liu¹Jun-hua Lu²Ying Zhao³Ying Lin³Li-qiu Ma³Ping Xue³Bao-yun Jin¹Bei-bei Li¹Zheng Zhou¹Xin-xin Huang¹Hong-jun Guan^1*

• ¹Department of Public Health, Mudanjiang Medical University, Mudanjiang, China
• ²Department of Nursing, Mudanjiang Medical University, Mudanjiang, China
• ³Hongqi Hospital Affiliated to Mudanjiang Medical University, Mudanjiang, China

Introduction: Ischemic stroke (IS) is a cerebrovascular disease that can be disabling and fatal, and there are limitations in the clinical treatment and prognosis of IS. It has been reported that changes in the expression profile of circRNAs have been found during injury in ischemic stroke, and circRNAs play an important role in the IS cascade response. However, the specific mechanisms involved in the pathogenesis of IS are not yet fully understood, and thus in-depth studies are needed.

Methods: In this study, one circRNA dataset (GSE161913), one miRNA dataset (GSE60319) and one mRNA dataset (GSE180470) were retrieved from the Gene Expression Omnibus (GEO) database and included, and the datasets were differentially expressed analyzed by GEO2R and easyGEO to get the DEcircRNA, DEmiRNA and DEmRNA, and DEmRNA was enriched using ImageGP, binding sites were predicted in the ENCORI database, respectively, and the competitive endogenous RNA (ceRNA) regulatory network was visualized by the cytoscape software, and then selected by MCC scoring in the cytoHubba plugin Hub genes. In addition, this study conducted a case–control study in which blood samples were collected from stroke patients and healthy medical examiners to validate the core network of ceRNAs constructed by biosignature analysis by real-time fluorescence quantitative qRT-PCR experiments.

Results: A total of 233 DEcircRNAs, 132 DEmiRNAs and 72 DEmRNAs were screened by bioinformatics analysis. circRNA-mediated ceRNA regulatory network was constructed, including 148 circRNAs, 43 miRNAs and 44 mRNAs. Finally, CLEC16A|miR-654-5p|RARA competitive endogenous regulatory axis was selected for validation by qRT-PCR, and the validation results were consistent with the bioinformatics analysis.

Discussion: In conclusion, the present study establishes a new axis of regulation associated with IS, providing new insights into the pathogenesis of IS.

1 Introduction

Stroke is a type of cerebrovascular disease in which a cerebral blood vessel suddenly ruptures or becomes blocked, resulting in insufficient blood supply to the brain and thus causing severe brain tissue damage and loss of neuronal function. It is one of the leading causes of mortality and morbidity worldwide and is a chronic non-communicable disease that poses a serious threat to the health of the global population. Ischemic stroke (IS) is one of the two main categories of stroke (1), commonly known as cerebral infarction, and refers to the occlusion of the arteries supplying the brain, which leads to necrosis of brain tissue and focal neurological deficits. Ischemic strokes occur every year, and most of them are in the elderly, and can be seriously disabling and fatal (2), causing a heavy medical burden on patients, families and society. According to the data published by the World Health Organization (WHO), the incidence of stroke is getting younger globally, which is a disturbing trend as people’s knowledge about stroke is increasing. Therefore, the prevention and treatment of ischemic stroke should be given sufficient attention to elucidate its pathogenesis as soon as possible and develop solutions to improve the quality of life of the population.

The current diagnosis of stroke relies on clinical symptoms and medical imaging techniques. The most commonly used diagnostic tools for IS are computed tomography (CT) and magnetic resonance (MR) (3). However, CT has poor sensitivity to early ischemic changes in the brain and most patients do not show typical imaging changes in the early stages of the disease, which may miss the optimal time for treatment. Restoration of cerebral blood flow supply is currently the main therapeutic strategy for acute cerebrovascular disease, and early re-establishment of cerebral blood supply by revascularization techniques of intravenous thrombolysis or arterial thrombolysis is the most effective clinical treatment for IS (4). However, there are limitations in the duration of treatment and the application of techniques, and most patients are left with irreversible brain tissue damage with the risk of hemorrhage and ischemia–reperfusion injury (5). Therefore, it is urgent to explore a rapid and accurate biomarker for early diagnosis and prediction of IS, and it is important to further elucidate the pathophysiological mechanisms of ischemic stroke and find timely and effective therapeutic targets.

In recent years, many studies have reported that changes in the expression profiles of noncoding RNAs (ncRNAs) were found during the injury process in ischemic stroke. miRNAs are a class of ubiquitous single-stranded non-coding RNAs that can specifically bind to messenger RNAs (mRNAs) through base complementary pairing, interfering with the translational process and thus inhibiting or altering protein synthesis. Numerous studies have shown that miRNAs, as important mediators of gene regulation, play important roles in IS cascade reactions. Translateral ventricular injection of miR-377 inhibitor in MCAO rats attenuates ischemic brain injury by promoting angiogenesis and inhibiting brain inflammation induced by pro-inflammatory factor release (6). miR-455-5p expression is downregulated in brain tissue and peripheral blood after cerebral ischemia/reperfusion (I/R), and overexpression of miR-455-5p reduces neuroinflammation through inhibition of CCR5 expression, thereby attenuating I/R-induced injury (7). Endothelial miR-15a/16–1 is a negative regulator of cerebral angiogenesis and neurological recovery after stroke (8).

Circular RNA (circRNA) is a unique structure produced by reverse splicing with a high degree of stability, interspecies conservation, and tissue expression specificity, making circRNA an ideal molecular biomarker for diagnosis (9). With the development of bioinformatics analysis methods, many scholars have identified differences in the expression of circRNAs in different organisms and tissues. In a study to identify differentially expressed circRNAs in blood samples from Acute IS patients and healthy controls by circRNA microarray, three circRNAs (circFUNDC1, circPDS5B, and circCDC14A) were up-regulated in AIS patients compared to healthy subjects, and the ROC curve showed an area under the curve (AUC) of 0.875, with a specificity of 91% and a sensitivity of 71.5%, indicating that these 3 circRNAs have diagnostic and prognostic value for AIS (10). The expression of circFOXP1 was significantly reduced in the peripheral blood of AIS patients, and overexpression of circFOXP1 could attenuate post-ischemic brain injury by regulating the STAT3/apoptosis signaling pathway (11). Studies have shown that circRNAs can act as miRNA sponges to regulate gene expression, interact with proteins to perform important biological functions, and encode proteins as translational templates. In recent years, the competitive endogenous RNA (ceRNA) mechanism of circRNAs has been extensively studied. Briefly, circRNAs chelate miRNAs through spongy competition and regulate the inhibitory effect of miRNAs on base complementary pairing at target sites in the untranslated region of messenger RNAs (mRNAs), which in turn regulates the expression of downstream target genes (12). A number of studies have identified a close association between circRNA-mediated ceRNA regulatory networks and ischemic stroke pathophysiological processes (13). A study demonstrated that the expression of circ-HECTD1 and tumor necrosis factor receptor-associated factor 3 (TRAF3) was significantly upregulated in ischemic brain tissues, whereas the expression of miR-133b was downregulated. Knockdown of circ-HECTD1 attenuated neuronal damage induced by cerebral ischemia by targeting binding to miR-133b and inhibiting TRAF3 expression, thereby inhibiting OGD-induced apoptosis and NF-κB activation (14). SNHG15 is upregulated in hypoxic–ischemic mice or cellular models, and inhibition of SNHG15 expression ameliorates ischemia-hypoxia-induced neuronal injury and microglial cell inflammation via the miR-302a-3p / STAT1 / NF-κB pathway (13). Han et al. (15) showed that CircHECTD1 levels were significantly increased in a transient middle cerebral artery occlusion (TMCAO) mouse stroke model, which was validated in plasma samples from AIS patients. By interfering with CircHECTD1 expression using an siRNA approach, MIR142 was released and accompanied by the downstream down-regulation of TIPARP expression, which resulted in the improvement of cerebral infarction by inhibiting astrocyte activation. In summary, circRNA is closely related to the pathophysiological process of ischemic stroke. As the abnormally expressed circRNA molecules are continuously mined for neuroprotective or deleterious effects on IS through the ceRNA regulatory pathway, the specific mechanisms of circRNA and stroke onset, progression and prognosis will be gradually clarified.

In this paper, we take ischemic stroke as an entry point to reveal the pathogenesis of ischemic cerebrovascular diseases from the perspective of genomics, so as to provide more valuable reference information for the clinic. In this study, we obtained circRNA, miRNA and mRNA expression datasets from the Gene Expression Omnibus (GEO) database, screened the differentially expressed genes and constructed the IS-associated ceRNA network, and then extracted a regulatory axis from them by combining with the Protein–protein interaction (PPI) network analysis, and then verified the gene expression levels through reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the results of the present study may provide a new idea for elucidating the potential mechanisms underlying the occurrence and development of ischemic stroke. The flow chart illustrating the steps of the whole analysis was shown in Figure 1.

More at link.