In our study, we accessed the possibility of circRNA polymorphisms as prognostic biomarkers for IS. To the best of our knowledge, our study is the first study to examine the role of circ-RNA in the recurrence and recovery of IS. Our findings identified that circ-STAT3 rs2293152 GG genotype were significantly associated with unfavorable functional outcome of IS, and rs2293152 could be served as prognostic biomarkers for IS patients. However, the other four SNPs were not associated with functional outcome of IS after 3 months. We also failed to find the association between all the circRNA polymorphisms and IS recurrence risk. Our study will provide novel perspectives for prognosis prediction and target gene-therapy for IS.
Recent studies have suggested that circRNAs might be novel diagnostic and prognostic biomarkers for the disease. Zuo et al. [20] found that three differentially expressed circRNAs (circFUNDC1, circPDS5B, and circCDC14A)in blood of IS patients through two stage studies. Subsequently, the study of Dong et al. [7] indicated that 521 differentially expressed circRNAs (373 increased and 148 circRNAs decreased) in the IS group compared with controls. CircRNA expression profiles were altered significantly in the PBMCs of IS patient and may be participate in the pathogenesis of IS. Meantime, recent association studies revealed that circRNAs polymorphisms conferred prognostic and susceptibility biomarkers for the disease. Burd et al. [21] suggested that the rs7341786 within 9p21 contribute to atherosclerotic vascular disease susceptibility through regulation of ANRIL splicing and circular ANRIL expression. In addition, Paraboschi et al. reported that hsa-circ_0043813 from the STAT3 gene was associated with multiple sclerosis risk and the genotype CC of rs2293152 could increase circ-STAT3 expression [16]. Moreover, Zhang and his colleges [17]. have found that rs25497 in circ-TUBB was associated with colorectal cancer risk in both Chinese and European populations by influencing the expression of ELF5 targeted to miR-4664-3p. Additionally, another study indicated that circ-FOXO3 rs12196996 at the gene flanking intron was associated with coronary artery disease risk in the Chinese Han population, which affected circ-FOXO3 expression, but not linear FOXO3 levels [22]. Furthermore, Guo et al. [23] found that circ-ITCH rs10485505 and rs4911154 were significantly associated with increased hepatocellular carcinoma risk. Until now, study about the circRNA polymorphisms and stroke prognosis is fewer.
Our study demonstrated that circ-STAT3 rs2293152 predicted functional outcome after stroke, carrying GG genotype exhibited worse outcomes 3 months post-stroke. We identified that the GG genotype was associated with increased risk of unfavorable outcome of stroke and that the CC + CG genotype was associated with a better outcome at 3 months. After adjustment for NIHSS score and other factors, the association for rs2293152 after 3 months outcome after IS was stronger, suggesting that rs2293152 is an independent risk factor for stroke recovery. However, no significant association between circ-DLGAP4 rs41274714, circ-TRAF2 rs10870141, circ-ITCH rs10485505, rs4911154 and short-term outcome of stroke was detected. Sub-group analysis revealed that the negative effect of rs2293152 GG genotype was greater in female, older patients and subjects with hypertension status. The results indicated the interaction of age, sex, blood pressure and rs2293152 enhanced the poor recovery of IS. There is a fact that risk factors for stroke recovery including hypertension, depression, atrial fibrillation were significantly more common in women [24], which may contribute the different risk among the gender. Moreover, individuals with older age are prone to have other chronic disease compared with youngers, which may influence stroke recovery. Additionally, all the circRNA polymorphisms were not correlated with a recurrent ischemic stroke risk.
The possible explanation of circ-STAT3 role in short-term prognosis might be as follows. First, the genetic variation at circ-STAT3 might influence the expression of STAT3 by functioning as miRNA sponge [25]. Second, we speculated that circ-STAT3 rs2293152 located at flanking intron may act as circ-eQTL and affect the circRNA biogenesis, which eventually influenced the expression level of circ-STAT3 [11]. This is consistent with Liu et al. study that genetic variants of circRNA within flanking intron region would influence circRNA expression [10]. Third, functional prediction revealed that rs2293152 was located at potential functional regions and might alter the binding affinity of regulatory motifs, which would influence circ-STAT3 expression. From the above evidence, we can speculate that the effects of circ-STAT3 rs2293152 on short-term prognosis of stroke seem to be mediated by regulating of circ-STAT3 expression. The increased expression of circ-STAT3 might promote the release of inflammatory cytokines and activate the inflammatory response. In addition, STAT3 may also regulated astrocyte activation through targeting of the JAK2/STAT3 pathway [26]. Astrocyte activation can aggravate inflammatory reactions and brain injury. Thus, circ-STAT3 may influence IS recovery by influence the neuro-inflammation processes after neural injury.
The highlights of this study were as follows. First, previous studies focused mainly on protein-coding genes, but our studies emphasized non-coding RNAs such as circRNAs. Our study is first and comprehensive study to demonstrate the potential role of circRNAs polymorphisms for functional outcome after stroke. Besides, NIHSS score was an important factor for the short-term outcome after stroke, which was took into account in our study. Thus, logistic regression was applied in our study to adjust the confound factors. Finally, we also did stratification analysis to find the interactions between rs2293152 and sex, age. However, there are limitations in our study. First, the sample size of our study was not large enough, further studies in different populations with larger sample are needed to validate the association between the circRNAs polymorphisms and functional outcome after stroke. Moreover, functional mechanisms of rs2293152 on the short-term prognosis after stroke are still not clear, which is needed to be clarified in the further study.
Our study demonstrated that circ-STAT3 rs2293152 GG genotype was associated with unfavorable outcomes 3 months after stroke. Moreover, rs2293152 of circ-STAT3 can also be used as the biomarker for predicting functional outcome after stroke. Further research is needed to explore the exact biological mechanism of these genetic variations on stroke recovery. A comprehensive understanding of genetic variants effect on stroke recovery is needed for setting up personalized therapeutic interventions after stroke.