miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population

Absstract Spleen tyrosine kinase (SYK) gene has been identified as novel susceptibility locus for ischaemic stroke (IS) previously. However, regulation of SYK gene remains unknown in IS. In this study, we aimed to identify miRNAs that might be involved in the development of IS by targeting SYK gene. miRNAs were firstly screened by bioinformatics predicting tool. The expression levels of SYK gene were detected by qRT‐PCR and western blotting, respectively, after miRNA transfection. Luciferase reporter assay was applied to investigate the direct binding between miRNAs and target gene. miRNA levels were detected by miRNA TaqMan assays in the blood cells of 270 IS patients and 270 control volunteers. Results suggest that SYK gene might be a direct target of miR‐129‐2‐3p. The blood level of miR‐129‐2‐3p was significantly lower in IS patients (P < 0.05), and negatively associated with the risk of IS (adjusted OR: 0.88; 95% CI: 0.80‐0.98; P = 0.021) by multivariable logistic regression analysis. The blood levels of SYK gene were significantly higher in IS patients, and miR‐129‐2‐3p expression was negatively correlated with mean platelet volume. In summary, our study suggests that miR‐129‐2‐3p might be involved in the pathogenesis of IS through interrupting SYK expression and the platelet function, and further investigation is needed to explore the underlying mechanism.

and thrombosis. 3,4 In addition to traditional risk factors, such as age, male sex, smoking, history of hypertension, diabetes mellitus and atrial fibrillation, amounting genetic risk factors have been identified in recent years by genome-wide association studies and exome sequencing. [5][6][7][8][9][10] We have previously identified several susceptibility loci for IS in a Chinese Han population. 11 Exome sequencing was used to screen susceptibility loci among 100 cases and 100 matched controls. Significant SNPs were further verified in up to 3554 participants from three hospital-based case-control studies. Ultimately, we identified two novel loci (rs17118 of XYLB gene located on chromosome 3p21, and rs10489177 of c1orf156 gene located on chromosome 1q24), which were associated with an increased risk of IS. Besides, we also found that the frequency of rs2290890 T allele of spleen tyrosine kinase (SYK) gene was associated with a decreased risk of IS (data not published). SYK gene, located on chromosome 9p22 and encoding a non-receptor type of protein-tyrosine kinase, that is, widely expressed in hematopoietic cells, is one of the two members of the Syk family (Syk and ZAP-70), and has been extensively studied as a critical regulator in the pathogenesis of cancer. 12 SYK gene polymorphisms have been also reported to be associated with the risk of non-Hodgkin lymphoma 13 and prostate cancer. 14 Recently, the role of SYK in vascular inflammation and atherosclerosis has attracted great interest. In the low-density lipoprotein receptor-deficient mice consuming a high-cholesterol diet supplemented with orally available SYK inhibitor fostamatinib, the atherosclerotic lesion size was reduced by 59%, the adhesion and migration of inflammatory cells were attenuated, and macrophage survival was limited, suggesting SYK inhibition as a potentially fruitful anti-inflammatory therapeutic strategy in atherosclerosis. 15 In addition, Bijli et al suggested that Syk signals downstream of PKCdelta to mediate thrombin induced ICAM-1 expression in endothelial cells by increasing transcriptional capacity of NF-kappaB via a mechanism that relies on tyrosine phosphorylation of RelA/p65. 16 According to the literature review and our previous findings, we supposed that the dysregulation of SYK expression might be crucial for the development of IS, an atherosclerosis-related disease. However, no direct evidence has been reported about the expression and regulation of SYK in IS.
miRNAs are a class of 22-nucleotide-long endogenous non-coding RNAs which participate in the post-transcriptional regulation of genes by binding to the 3′ untranslated region (3′UTR). miRNAs are well recognized as key regulators in the pathological events of IS, including atherosclerosis, 17 hyperlipidemia, 18 hypertension 19 and plaque rupture. 20 Inspired by the clinical application of oral SYK inhibitors as previously indicated, 15 we further have been suggested that the decrease in SYK expression might be partially regulated by miRNA. To figure out the miRNAs that might target SYK expression and the miRNAs with potential clinical value as biomarker for IS, we firstly screened for miRNAs by the bioinformatics predicting tool  For the detection of SYK gene expression, the total RNA was isolated from 500 μL of whole blood cells/sample using Trizol LS reagent (Invitrogen) according to the manufacturer's protocol.

| Target prediction and miRNA selection
The miRNAs that might target SYK gene were firstly predicted by in silico analysis using a bioinformatics tool: TargetScan (http://www.ta rgetscan.org/vert_71/). We previously explored the miRNA expression profiles of 25 IS patients and 25 control volunteers using miRNA microarray and found that 455 miRNAs were differentially expressed (unpublished data, P < 0.05). Ultimately, a total of 20 differentially expressed miRNAs with the most negative cumulative weighted context++ score by TargetScan Human 7.1 were selected for further investigation as shown in Table S1.

| Statistical analysis
The normal distribution of data was tested using the one-sample Kolmogorov-Smirnov test. Continuous variables were expressed as the mean ± SD or median (25th-75th quartile), and categorical variables were expressed as frequency. Differences in clinical characteristics HUANG ET AL.
| 169 between cases and controls were examined by the chi-squared test for categorical variables, by Student's t-test for normally distributed data, or by the Mann-Whitney U test for skewed data. The difference in miR-129-2-3p expression levels between cases and controls, and SYK mRNA or protein levels between cells with different treatment was examined by Student's t-test. Comparison of the relative luciferase activities (RLAs) between cell groups were examined by one-way ANOVA. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the association between miR-129-2-3p and IS using the multivariate logistic regression model after adjusting for conventional risk factors, including age, sex, smoking, drinking, hypertension and diabetes mellitus. Correlations between miRNA expression and clinical variables were analysed using the Pearson or Spearman correlation test. All statistical were performed with SPSS 16.0 software (Statistical Package for the Social Sciences, Chicago, IL, USA). A value of P < 0.05 was considered significant (two-tailed).

| Regulation of SYK expression by miR-129-2-3p
The microarray data and the cumulative weighted context++ scores of the 20 miRNAs were shown in Table S1. Among the miRNAs, after transfection of miR-129-2-3p mimics for 48 hours, the mRNA level of SYK gene was both down-regulated in THP-1 and U937 cells, with a decreased level to 90% (THP-1) and 55% (U937) of the cells, respectively, compared with the mimic control ( Figure 1A). However, the mRNA levels of SYK gene showed no alteration in cells transfected with other miRNA mimics ( Figure 1A and B). Afterwards, we evaluated the change in SYK protein levels after miR-129-2-3p transfection.
Results suggest that cells transfected with miR-129-2-3p mimics showed decreased protein levels of SYK in both THP-1 (to a 50% extent) and U937 cells (to a 40% extent), as shown in Figure 2A     The motive that initiates the conduction of this study was the association that we found between SYK gene polymorphism and the risk of IS, which was identified by exome sequencing in one of our previous studies, 11  Besides, MPV can even provide some information about the prognosis of stroke patients. 35 In this study, we found that the MPV levels of IS patients were significantly higher than the control volunteers and the decreased level of miR-129-2-3p was negatively correlated with MPV in the stroke patients ( Figure 5). Several platelet functions rely on SYK signaling. GpVI, the major collagen receptor of platelets, is an FcRγ-associated receptor closely related to FcαRs, which may explain that GpVI, like other FcR family members, signals through  The expression levels of SYK gene were detected in the whole blood of 60 ischaemic stroke patients and 60 control volunteers. The relative expression levels were normalized to beta-actin and then logtransformed. The whiskers of the plots represent the 5-95 percentiles. Comparison of gene expression between the two groups of cohorts was analysed by the Student's t test both exist in stroke genetics, 40 accumulating evidence to explore the functional SNPs associated with stroke have been reported recently, by means of eQTLs. 41,42 Inspired by the above studies, we will further combine in silico and experimental approaches to explore the functional SNPs of SYK gene, which might improve our understanding of disease mechanism in stroke and likely provide the field with novel targets and pathways for intervention.

| SYK expression in the blood cells of IS
In summary, this study suggests that the blood cell level of miR-129-2-3p is significantly lower in IS patients and associated with the occurrence of IS. SYK gene might be a direct target of miR-129-2-3p, and the expression levels of SYK gene were significantly higher in IS patients compared with control volunteers. In addition, miR-129-2-3p expression was negatively correlated with the platelet parameter MPV. Further investigations are needed to explore the biological relevance of our findings.

ACKNOWLEDGEMENTS
We are particularly grateful to all the ischaemic stroke patients and volunteers who participated in this study and to the medical personnel of People's Hospital of Shenzhen for their kind assistance in collecting questionnaires and blood samples.

CONF LICT OF I NTEREST
The authors confirm that there are no conflicts of interest.