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Long non‐coding RNA UCA1 correlates with elevated disease severity, Th17 cell proportion, inflammatory cytokines, and worse prognosis in acute ischemic stroke patients

Abstract Background This study aimed to explore the association of long non‐coding RNA urothelial carcinoma‐associated 1 (lncRNA UCA1) expression with disease severity, inflammation, and prognosis in acute ischemic stroke (AIS) patients. Methods The lncRNA UCA1 expression of blood CD4+ T cells from 160 first‐episode AIS patients and 160 non‐AIS patients with high‐stroke‐risk factors (as controls) was detected by reverse transcription quantitative polymerase chain reaction. For AIS patients, interleukin (IL)‐6, IL‐17, and intracellular adhesion molecule‐1 (ICAM1) were determined by enzyme‐linked immunosorbent assay; Th17 cell ratio in CD4+ T cells was detected by flow cytometry. Their follow‐up data were recorded up to 36 months, recurrence of stroke or death. The recurrence‐free survival (RFS) analysis was assessed according to the follow‐up data. Results LncRNA UCA1 expression was higher in AIS patients compared to controls (p < 0.001), and it was positively correlated to national institute of health stroke scale score (r = 0.436, p < 0.001), Th17 cell ratio (r = 0.398, p < 0.001), IL‐6 (r = 0.204, p = 0.010), IL‐17 (r = 0.326, p < 0.001), and ICAM1 (r = 0.276, p < 0.001) in AIS patients. Regarding prognosis, lncRNA UCA1 expression was elevated in 2‐year recurrence/death AIS patients compared to those patients without recurrence or death within 2 years (p = 0.033), also increased in 3‐year recurrence/death AIS patients compared to those patients without recurrence or death within 3 years (p = 0.008). Furthermore, high lncRNA UCA1 expression was associated with worse accumulating RFS (p = 0.017) in AIS patients. Conclusion LncRNA UCA1 might sever as a candidate prognostic biomarker in AIS patients, suggesting its potency for AIS management.


| INTRODUC TI ON
Acute ischemic stroke (AIS) is a cerebrovascular disease characterized by blockage of the cranial vasculature, which leads to insufficient oxygen and nutrients supply to the affected brain area. 1 Notably, it is the third most common cause of death for elderly people with prevalence rate of 36.8% for patients above 60 years in high-income countries. 2 Regarding the current treatment for AIS, it involves thrombolysis and embolectomy surgery to resolve the thrombosis and re-establish normal blood flow in the vessels. 3 However, AIS patients tend to develop restenosis and recurrence due to prolonged ischemic period and profound affected brain area, which results in poor prognosis and reduced quality of life in them. 3 Therefore, finding a potential and novel factor to monitor disease progression and predict prognosis in AIS patients is necessary.
Long non-coding RNA (lncRNA) is a type of RNA whose length over 200 nucleotides with less coding function, which has been reported to be highly involved in angiogenesis and inflammation, and some of them have been identified as key regulators in carido-cerebrovasular pathophysiology, especially in the stroke. [4][5][6] As a commonly studied lncRNA, lncRNA urothelial carcinoma-associated 1 (lncRNA UCA1) is initially found in bladder carcinoma and serves as a tumor enhancer in various types of cancer (such as colorectal cancer, gastric cancer, and hepatocellular carcinoma). 7-10 More interestingly, accumulating evidence suggests the crucial role of lncRNA UCA1 in various cadio-cerebovascular diseases. [11][12][13] For instance, lncRNA UCA1 regulates microRNA (miR)-18a expression to promote hypoxia injury and neural cell apoptosis in the cell model of cerebral ischemia. 14 Moreover, small RNA interference targeting IncRNA UCA1 decreases pro-inflammatory cytokines (such as IL-6 and ICAM1) at gene level and prevents their induction in lipopolysaccharide (LPS) treated human dermal microvascular endothelial cells, indicating the correlation of lncRNA UCA1 with inflammatory cytokines. 11 Besides, inflammation plays an important role in the pathogenesis of AIS since inflammation leads to unstable atherosclerotic plaque with higher risk of rupture. In the clinical practice, lncRNA UCA1 expression is upregulated in acute myocardial infraction (AMI) patients, which shares similar pathophysiological mechanism as AIS since both of these two diseases characterized by the blockage of blood vessels due to atherosclerosis plaque formation. 12 In addition, we performed a preliminary research in a smaller sample size and discovered that lncRNA UCA1 expression was upregulated in tested AIS patients. Based on those abovementioned evidence, we hypothesized that lncRNA UCA1 may play a critical role in AIS. However, no relevant research has been conducted yet. Therefore, we conducted this study and aimed to investigate the correlation of lncRNA UCA1 expression with disease severity, inflammation, and prognosis in AIS patients.

| Study population
This study consecutively recruited 160 first-episode AIS patients treated in our hospital between May 2015 and July 2017. AIS patients were eligible for inclusion if they met the following criteria: (i) confirmed diagnosis of AIS; (ii) admitted to our hospital within 12 hours of stroke onset; (iii) age ≥18 years old; and (iv) no intracranial hemorrhage was found. Patients with any of following conditions were excluded from study: (i) had medical history of stroke before study recruitment; (ii) known neurological diseases (eg, glioma); (iii) history of severe infections or systemic inflammatory disease; (iv) treatment with immunosuppressive drugs with 3 months before enrollment; (v) history of hematological malignancies (eg, leukemia) or solid tumors; and (vi) female patients in pregnant. In addition, 160 non-AIS patients with high-stroke-risk factors in our hospital were recruited as study controls during the same period. The eligible criteria for controls were as

| AIS patients' characteristics and blood sample collection
After recruitment, basic characteristics of AIS patients were documented for analysis, including demographic information and underlying disease. National Institute of Health stroke scale (NIHSS) score was evaluated on the day of admission, which was used for disease severity assessment. 16 Peripheral blood samples of AIS patients were also collected on the day of admission, which were divided into two parts: one was processed by centrifugalizing to separate serum samples for measurement of interleukin-6 (IL-6), interleukin-17 (IL-17), and intercellular cell adhesion molecule-1 (ICAM1); the other was treated with Dynabeads™ FlowComp™ Human CD4 Kit (Invitrogen) to isolate CD4 + T cells from blood samples according to the manufacturer's instructions. After isolating CD4 + T cells, the Th17 cell ratio in the CD4 + T cells was measured immediately by flow cytometry. The total RNA in CD4 + T cells was extracted using TRIzol™ Reagent (Invitrogen) and stored at −80℃ for lncRNA UCA1 detection.

| Controls' characteristics and blood sample collection
The characteristics of controls (demographic information and underlying disease) were also collected after enrollment. The blood samples of controls were collected to isolate the CD4 + T cells as same as that in AIS patients. The total RNA in CD4 + T cells was extracted using TRIzol™ Reagent (Invitrogen) and stored at −80℃ for lncRNA UCA1 expression detection as well.

| LncRNA UCA1 expression detection
For the AIS patients and controls, the freeze-stored total RNA from CD4 + T cells was used to detect lncRNA UCA1 expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR). In brief, iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, California, USA) was used for reverse transcription to convert RNA into complementary DNA. Furthermore, the THUNDERBIRD ® SYBR ® qPCR Mix (Toyobo) was applied for qPCR reaction. The entire thermal cycles were conducted as follows: 95℃ for 5 minutes, followed by 40 cycles of 95℃ for 5 seconds and 61℃ for 30 seconds. The lncRNA UCA1 expression was calculated by 2 −ΔΔCt method using GADPH as internal reference.
Primers were designed according to a previous study 11

| RFS evaluation
All AIS patients were followed up every 3 to 6 months through clinic visits or telephone contact, and the follow-up was continued until the completion of 36-month visit, the recurrence of stroke, or death.
Recurrence-free survival (RFS) was assessed according to the follow-up data. The RFS was defined as the duration from hospitalization to stroke recurrence or death. The patients who lost follow-up were censored at the last visit date.

| Statistical analysis
Statistical analysis and graphs construction were performed using SPSS 24.0 software (IBM) and GraphPad Prism 7.01 software (GraphPad Software lnc.). Comparison between two groups was determined by Student's t test, Wilcoxon rank sum test, or chisquare test. Correlation analysis was determined by Spearman's rank correlation test. Receiver-operating characteristic (ROC) curve and the area under the curve (AUC) with 95% confidence interval (CI) were used to assess the ability of lncRNA UCA1 in predicting recurrence/death risk. RFS was displayed using Kaplan-Meier curve, and the comparison of RFS between two groups was analyzed by log-rank test. p Value <0.05 was considered statistically significant.

| Correlation of lncRNA UCA1 expression with disease severity and inflammation in AIS patients
As to disease severity, lncRNA UCA1 expression was positively correlated to NIHSS score (r = 0.436, p < 0.001) (Figure 3).

| Correlation of lncRNA UCA1 expression with prognosis in AIS patients
No difference of lncRNA UCA1 expression was found between 1-year recurrence/death AIS patients and those patients without recurrence

| DISCUSS ION
In the present study, we discovered that (a) lncRNA UCA1 expression was increased in AIS patients compared to controls, (b) lncRNA UCA1 expression was positively correlated to NIHSS score and inflammation (including Th17 cell proportion, IL-6, IL-17, and ICAM1) in AIS patients, and (c) increased lncRNA UCA1 expression was related to poor prognosis in AIS patients.
Accumulating evidence suggests that lncRNA UCA1 may play an important role in multiple neurological diseases. For example, elevated lncRNA UCA1 expression is found in both mice brain tissue and cell model of PD. 17 In addition, lncRNA UCA1 expression also has been reported to be reduced in epilepsy mice brain tissue. 18 Apart from the dysregulation of lncRNA UCA1 in various neurological diseases, several studies also explore the mechanism of lncRNA UCA1 underlying neurological diseases. For example, abnormal expression of lncRNA UCA1 promotes miR-18a expression leading to hypoxia injury and neuronal apoptosis in cell model of cerebral ischemia. 14 Moreover, lncRNA UCA1 promotes PI3 K/AKT signaling pathway to enhance oxidative stress and inflammation in mice model of PD. 13 Furthermore, lncRNA UCA1 promotes apoptosis-related pathway (such as caspase-3 activity) and enhances neuronal apoptosis in cell model of PD. 17 Taken together, lncRNA UCA1 is obviously involved in the pathophysiology of neurological diseases. More interestingly, lncRNA UCA1 has been identified as a potential factor in cardio-cerebrovascular diseases. For instance, lncRNA UCA1 expression is increased in AMI patients compared to control subjects. 12 Another study demonstrates that lncRNA UCA1 expression varies in AMI patients which is reduced initially, then increased in Day 3 after AMI occurrence. 19 Besides, lncRNA UCA1 regulates pro-inflammatory cytokines production (such as IL-6 and ICAM1) in LPS treated endothelial cells. 11 Moreover, ln-cRNA UCA1 expression is elevated, which also promotes neuron injury via miR-18a/SOX6 axis in PC12 cellular cerebral ischemia model. 14 Since lncRNA UCA1 is under heavy investigation in both cardio-cerebrovascular and neurological diseases, also previous studies suggest that lncRNA UCA1 participates in endothelial cell inflammation process and cerebral ischemia related injury, thus we hypothesized that lncRNA UCA1 might also play a crucial role in AIS patients. However, no clinical research regarding the role of lncRNA UCA1 in AIS patients has been performed. In the present study, we discovered that lncRNA UCA1 expression was elevated n AIS patients compared to controls. The possible reasons were as follows: (a) Abnormal IncRNA UCA1 expression might regulate miR-18a expression, thereby leaded to hypoxia injury and neuronal apoptosis in cerebral ischemia. 14 Thus, abnormal lncRNA UCA1 expression was found in AIS patients. (b) lncRNA UCA1 might regulate pro-inflammatory cytokines expression to enhance local inflammatory response. 11 Meanwhile, accumulating evidence suggested that local inflammatory response assisted in the pathogenesis of ischemic events. 5,6 Therefore, elevated lncRNA UCA1 expression was correlated with higher AIS risk.

TA B L E 1 Comparison of clinical characteristics
The correlation of lncRNA UCA1 with inflammation in cardio-cerebrovascular disease has been explored. For instance, an interesting study finds that lncRNA UCA1 regulates IL-6 and ICAM1 in LPS treated endothelial cells. 11 In our study, we found that lncRNA Limited study reports the prognostic power of lncRNA UCA1 in cardio-cerebrovascular diseases. In order to fill this vacancy, we conducted this study and found that lncRNA UCA1 showed a predictive value for 2-year and 3-year recurrence/death risk; also, its high level was related to worse accumulating RFS in AIS patients. an independent risk factor for AIS recurrence would be conducted in the following studies. Finally, the underlying mechanism in the effect of lncRNA UCA1 on Th17 cell ratio and inflammatory cytokines (including IL-6, IL-7, and ICAM1) was not explored; thus, further study could investigate this aspect.
In conclusion, lncRNA UCA1 is correlated with elevated disease severity, Th17 cell proportion, inflammatory cytokines, and worse prognosis in AIS patients.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.