The clinical value of long noncoding RNA GAS5 in acute ischemic stroke: Correlation with disease risk, inflammation, severity, and risk of recurrence

Abstract Background Long noncoding RNA growth arrest‐specific 5 (lnc‐GAS5) is involved in the pathophysiology of acute ischemic stroke (AIS) by regulating vascular stenosis, inflammation, and neurocyte apoptosis. This study aimed to explore the clinical value of lnc‐GAS5 in patients with AIS. Methods Plasma samples were collected from 120 patients with AIS at admission and 60 controls after enrollment, and lnc‐GAS5 expression in the plasma of all participants was assessed by reverse transcription quantitative polymerase chain reaction. In patients with AIS, disease severity was evaluated using National Institute of Health Stroke Scale (NIHSS) score, and plasma inflammatory cytokine levels were measured by enzyme‐linked immunosorbent assay. Recurrence‐free survival (RFS) was calculated during a 36‐month follow‐up period. Results Lnc‐GAS5 expression levels were higher in patients with AIS than in the controls (p < 0.001), and it had the potential to discriminate the controls from patients with AIS (area under the curve: 0.893, 95% confidence interval: 0.849–0.938). In patients with AIS, elevated lnc‐GAS5 levels were positively correlated with NIHSS score (r = 0.397, p < 0.001), diabetes mellitus (p = 0.046), and higher levels of tumor necrosis factor alpha (TNF‐α; r = 0.374, p < 0.001), interleukin‐6 (IL‐6; r = 0.223, p < 0.001), and interleukin‐17A (IL‐17A; r = 0.222, p = 0.015). The expression levels of lnc‐GAS5 were also negatively correlated with the levels of interleukin‐10 (IL‐10; r = −0.350, p < 0.001) and RFS (p = 0.036). Conclusion Lnc‐GAS5 is correlated with higher susceptibility to AIS, inflammation, and severity, and can predict an increased risk of AIS recurrence, indicating that monitoring of lnc‐GAS5 might improve the management of AIS.


| INTRODUC TI ON
Acute ischemic stroke (AIS) is a cerebrovascular disease induced by arterial stenosis or occlusion and is one of the leading causes of neurological morbidity and mortality worldwide. [1][2][3] The risk factors for AIS include diabetes mellitus, carotid atherosclerosis, and dyslipidemia, among others. [1][2][3][4] The main treatment strategies for AIS include thrombolytic therapy, anticoagulation therapy, and neuroprotective therapy. However, patients with AIS still face long-term adverse outcomes, such as cognitive and functional impairment. 5,6 Furthermore, until present, several studies have explored the potential biomarkers of AIS; however, recurrence risk and mortality of AIS are still elevating. [7][8][9] Thus, reliable biomarkers to monitor disease risk, severity, and prognosis are crucial to improve the management of AIS.

| Study participants
From February 2016 to December 2017, 120 first-episode AIS patients treated in our hospital were consecutively recruited for this prospective study. Patients who met the following criteria were eligible for enrollment: (a) newly diagnosed with AIS according to the American Stroke Association guidelines 19 ; (b) age >18 years; (c) with the occurrence of AIS within 12 h; (d) without intracranial hemorrhage; and (e) willing to be followed up regularly. The exclusion criteria were: (a) concomitant inflammatory disease or immune system disease; (b) presenting with active infections; (c) history of treatment with immunosuppressive agents within 3 months; (d) history of cancer or malignancy; and (e) pregnant or lactating. During the same period, 60 subjects who were at high risk of stroke were also enrolled as controls, and all of them had at least two of the following risk factors: current smoking, hypertension, hyperlipidemia, hyperuricemia, diabetes mellitus, and chronic kidney disease (CKD). The controls were also excluded from the study if they met any of the exclusion criteria for patients with AIS or had a history of stroke. Additionally, to eliminate age and sex as potential sources of bias, the age range of the controls was 55-80 years old, and the sex ratio was 3:2 (males to females).
This study was approved by the institutional review board, and the signed informed consents were collected from all participants or their statutory guardians.

| Data and sample collection
After enrollment, the clinical characteristics of the patients with AIS and controls were documented, including age, sex, body mass index (BMI), and risk factors for stroke. The National Institutes of Health Stroke Scale (NIHSS) scores of the patients with AIS were collected within 24 h after admission to assess disease severity. Peripheral blood (PB) samples were collected from patients with AIS on the day of admission and from the controls on the day of enrollment, and plasma was isolated for subsequent analyses.

| Lnc-GAS5 expression determination
The expression levels of lnc-GAS5 in the plasma of all study participants were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). In brief, total RNA was extracted using the QIAamp RNA Blood Mini Kit (Qiagen) and reverse transcribed using the iScript™ cDNA Synthesis Kit (with random primers) (Bio-Rad). qPCR was performed using SYBR ® Green Realtime PCR Master Mix (Toyobo). Relative lnc-GAS5 expression was calculated using the 2 −ΔΔC t method, and GAPDH was used as an internal reference. The primers used for qPCR were described in a previous study. 20

| Assessment of recurrence-free survival (RFS)
All AIS patients were followed up continuously until stroke recurrence, death, or completion of the 36-month follow-up. The final follow-up date was December 29, 2020. RFS was calculated based on the follow-up data. Patients who were lost to follow-up for any reason were censored at the last visit.  Table 1.

| Comparison of lnc-GAS5 levels between patients with AIS and controls
The optimal cut off for lnc-GAS5 expression was 1.865, with a sensitivity of 0.692 and specificity of 0.933 ( Figure 1B).

| Correlation of lnc-GAS5 with RFS in patients with AIS
To explore the correlation between lnc-GAS5 and the prognosis of patients with AIS, patients with AIS were followed up for 36 months, which revealed that elevated lnc-GAS5 was correlated with shorter RFS (p = 0.036), and RFS was significantly shorter in patients with lnc-GAS5 levels in Q4 than in patients with lnc-GAS5 levels in Q1 (p = 0.010; Figure 4).

| DISCUSS ION
In this study, we made several interesting observations: (1) lnc-GAS5 expression was higher in patients with AIS than in the controls and was a good predictor of AIS risk; (2) lnc-GAS5 was positively correlated with NIHSS score and pro-inflammatory cytokine expression (TNFα, IL-6, and IL-17A), and was negatively correlated with antiinflammatory cytokine (IL-10) in patients with AIS; and (3) lnc-GAS5 was negatively associated with RFS in patients with AIS.
Regarding the role of lnc-GAS5 in the risk of cardiocerebrovascular disease, a previous study showed that lnc-GAS5 was obviously elevated in the plaques of atherosclerosis patients when compared to the levels in healthy populations. 21 However, information on the correlation between lnc-GAS5 and AIS risk is limited. Thus, we compared the expression of lnc-GAS5 between AIS patients and healthy controls and used ROC curve analysis to estimate the predictive value of lnc-GAS5 for AIS risk, which revealed that lnc-GAS5 levels were higher in patients with AIS than in the controls, and lnc-GAS5 had good predictive value for AIS risk. A possible explanation for this might be that: (1) increased lnc-GAS5promoted apoptosis of vascular endothelial cells, resulting in arterial stenosis, which might play a role in the pathogenesis of AIS 15,18 ; (2) elevated lnc-GAS5 could modulate enhancer of zeste homolog 2 (EZH2)-mediated adenosine triphosphate-binding cassette transporter A1 transcription to accelerate the progression of atherosclerosis, which might increase the risk of AIS 15,22 ; (3) lnc-GAS5 might promote the formation of thrombosis, which could consequently accelerate AIS risk. 4,23 These data suggest that lnc-GAS5 might be useful as a novel biomarker of AIS risk. We also found that elevated lnc-GAS5 was correlated with the prevalence of diabetes mellitus in patients with AIS, suggesting that an increase in lnc-GAS5 might promoted the progression of diabetes mellitus, 24 consequently, diabetes could accelerate vascular aging, which might lead to the development of AIS. 25 The correlation of lnc-GAS5 with disease severity or inflammation in AIS has been rarely reported; thus, to explore this issue, for patients with AIS, the NIHSS score were collected to   This study has some limitations: (1) the follow-up duration was relatively short, and the correlation between lnc-GAS5 and the long-term prognosis of AIS patients could be investigated in a future study; (2) the mechanism by which lnc-GAS5 through its targets is involved in the pathogenesis and progression of AIS was not investigated, and thus could be explored in future studies; (3)

| CON CLUS ION
In conclusion, lnc-GAS5 is correlated with higher susceptibility to AIS and greater inflammation and severity and could predict the risk of recurrence to some extent, indicating that monitoring of lnc-GAS5 might improve the management of AIS.

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