The Diagnostic Value of Whole Blood lncRNA NR_120420 for Acute Ischemic Stroke

Objective Current clinical practice based on CT or multimodal images to diagnose ischemic stroke always led to substantial treatment delay. We perform this study to explore possible circulating lncRNA biomarker to help promptly diagnose the disease. Methods We used microarray to identify the differentially expressed lncRNAs in the peripheral whole blood between AIS patients and controls and verified the results by quantitative polymerase chain reaction (qPCR). Multivariate logistic regressions were performed to determinate the lncRNAs independently associated with AIS occurrence. The ROC curve was used to detect the diagnostic accuracy of candidate lncRNAs in AIS and AIS subtypes, which was classified according to the Oxford Community Stroke Project (OCSP) criteria. Results The microarray analysis screened out 5686 differentially expressed lncRNAs. Among the nine selected lncRNAs verified by qPCR, NR_120420 (OR 1.29, 95% CI 1.02-1.65, P = 0.037) was found independently associated with AIS after balancing patient baseline characteristics. The receiver operating characteristic (ROC) analysis concerning NR_120420 in total anterior circulation infarction subgroup showed that the area under the curve was 0.86 (95% CI: 0.73-0.99, P = 0.003), and at the optimal cutoff point of 1.93, the sensitivity and specificity reached 85.7% and 84.6%, respectively. Conclusion Our study indicated that NR_120420 could predict the total anterior circulation infarction with high sensitivity and specificity and could be potentially used as a biomarker for total anterior circulation infarction in AIS patients.


Introduction
Nowadays, thrombolysis and thrombectomy become standard treatment of acute ischemic strokes (AIS) [1][2][3][4][5]. However, patients' outcomes are closely related to onset to reperfusion time. Current clinical practice based on CT or multimodal images to diagnose the diseases always led to substantial treatment delay, i.e., patients with suspicious stroke have to be sent to hospital and perform CT before initiating such reperfusion treatment. Attempts for faster and more convenient diagnosis are needed to shorten the treatment delay and improve patient outcome.
It is speculated that, in AIS, many molecules and proteins released from injured or dead brain cells may escape from the brain via the disrupted blood-brain barrier and enter the blood flow, which may provide a new approach for the prompt diagnosis of AIS [6][7][8][9]. In recent years, the long noncoding RNAs (lncRNAs) have been used in the diagnosis of many diseases [10][11][12][13]. lncRNAs are a subtype of noncoding RNAs (ncRNAs) that make up the largest portion of the transcriptome [14] and are involved in many cellular processes after stroke including hypoxia, excitotoxicity, inflammation, oxidative stress, and apoptosis [15]. In such circumstance, some circulating lncRNAs may play important roles in early diagnosis of the AIS [16]. However, such studies are still limited.
In this study, we identified differentially expressed lncRNAs in the peripheral whole blood from AIS patients using microarray and verified them by quantitative polymerase chain reaction (qPCR); we aimed to explore the relationship between circulating lncRNA expression and the classification of AIS.

Materials and Methods
2.1. Patients. The patients with AIS admitted to the Neurovascular Center of Shanghai Changhai Hospital between January 2016 and November 2016 were enrolled in the study. The written informed consent was obtained from these patients. They were selected following the inclusion and exclusion criteria. Inclusion criteria were as follows: time duration from the onset of stroke to confirmation was less than 6 hours. Exclusion criteria were as follows: immune disease, trauma, organ failure, tumor, and infection. The control (CTRL) population was made up of healthy volunteers. The study was approved by the Medical Ethics Committee of Shanghai Changhai Hospital, and all the experiment procedures were carried out according to the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Blood
Collection and RNA Extraction. The peripheral whole blood of AIS patients and CTRLs was collected and added into PAXgene Blood RNA Tubes (BD, New York, America). Subsequently, the blood collection tube would be rapidly transferred to a refrigerator whose temperature was set at -80°C. Therefore, the total RNA was extracted with a PAXgene Blood miRNA Kit (Qiagen, Nordrhein-Westfalen, Germany) from the peripheral whole blood according to the manufacturer's instructions. The clinical data of AIS patients and CTRLs was obtained through clinical or outpatient follow-up.
2.3. lncRNA Microarray Hybridization. The Agilent Human lncRNA microarray (4 * 180K V5, Design ID: 076500) was used in this experiment. Total RNA was transcribed into double-strand cDNA and then synthesized into cRNA and labeled with Cyanine-3-CTP. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies, CA, USA). Lastly, Feature Extraction software (version 10.7.1.1, Agilent Technologies, CA, USA) was used to analyze array images to get raw data and Gene-Spring (version 13.1, Agilent Technologies, CA, USA) was employed to finish the basic analysis with the raw data. To begin with, the raw data was normalized with the quantile algorithm. Differentially expressed genes or lncRNAs with an arbitrary fold change (FC) (≥2) and a P value (<0.05) were selected.

Quantitative PCR.
A two-step reaction process was used for qPCR (reverse transcription (RT) PCR and real-time PCR). PrimeScript™ RT reagent Kit with gDNA Eraser (Takara-Bio, Shiga, Japan) was used in the first step because it can eliminate genomic DNA. QuantiNova SYBR Green PCR Kit (Qiagen, Nordrhein-Westfalen, Germany) was used in the second step. At the end of the PCR cycles, the sequencing work was performed by a 3730XL sequencer (Applied Biosystems, California, CA, USA) to validate the specific generation of the expected PCR product. The cycle threshold (Ct) was defined as the cycle number when the fluorescence has just exceeded the given threshold. The operators who detected the lncRNAs in blood were blinded to clinical data of the samples. Relative expression levels of lncRNAs were normalized to β-actin and were calculated by the 2 -ΔΔCT method in triplicate.

Correlation
Analysis between lncRNAs and Stroke. Multivariate logistic regressions were performed to evaluate the relationship among candidate lncRNA expression, clinical features, and AIS status. We used ROC curves to clarify the diagnostic accuracy of candidate lncRNAs for AIS.

Correlation between Specific lncRNAs and Stroke
Classification. AIS patients were classified according to the OCSP criteria by two independent examiners, namely, total anterior circulation infarct (TACI), partial anterior circulation infarct (PACI), posterior circulation infarct (POCI), or lacunar infarct (LACI), based on their maximum neurological defects [17]. The expression levels of specific lncRNA between AIS patients in different classifications and CTRLs were analyzed statistically.

Statistical Analysis.
A χ 2 test was used to compare the qualitative data. One-way analysis of variance (ANOVA) was applied when the quantitative data could follow the normal distribution (the Shapiro-Wilk test, α = 0:1) and the homogeneity of variance (the Levene test). The Mann-Whitney test or the Kruskal-Wallis test was performed when the quantitative data did not fit the normal distribution or the homogeneity of variance. All statistical calculations were performed with the SPSS software 21.0. The final data was expressed as mean ± standard deviation (SD), and a value of P < 0:05 was considered as significant.

Microarray Analysis
Results. The expression levels of lncRNAs and mRNAs in blood were detected by microarray technology. As a result, 5686 lncRNAs and 1990 mRNAs with a fold change ≥ 2:0 in the expression profile were identified as the differentially expressed genes. A clustering heat map (Figure 1(a)) exhibited distinguishable expressed lncRNAs between AIS patients and CTRLs.

NR_120420 Had High Specificity and Sensitivity in TACI Patients.
A receiver operating characteristic (ROC) curve was drawn to detect the diagnostic accuracy of lncRNAs as biomarkers for AIS (Figure 2(b), left). The area under the curve (AUC) of NR_120420 was 0.65 (95% CI: 0.53-0.78, P = 0:019). At the optimal cutoff point (0.63), the sensitivity and specificity were 53.8% and 76.9%, respectively.
We further examined the expression levels of NR_ 120420 in different AIS subtypes (Figure 3). Compared to CTRLs, its expression was increased in TACI and PACI subtypes and decreased in the POCI subtype. The differences were significant only in the TACI subtype compared to CTRLs. A ROC curve (Figure 2(b), right) concerning NR_ 120420 in TACI patients showed the AUC reached 0.86 (95% CI: 0.73-0.99, P = 0:003). At the optimal cutoff point of 1.93, the sensitivity and specificity were 85.7% and 84.6%, respectively.

Discussion
In this study, we identified one specific circulating lncRNA (NR_120420) that exhibited significantly higher in the AIS patients, and the validation via qPCR showed NR_120420 has a high sensitivity and specificity in the diagnosis of total anterior circulation infarction.
By using microarray and qPCR, we successfully identified two circulating lncRNAs (NR_120420 and lnc-GCH1-2:3) significantly increased in AIS patients in the beginning. However, lnc-GCH1-2:3 was not found independently associated with the occurrence of AIS after balancing with patient baseline characteristics, which indicated lnc-GCH1-2:3 may be more likely associated with such baseline characteristics.  Figure 3: Comparison of NR_120420 expression in AIS patients with different OCSP classification to the CTRL group. The relative expression levels of NR_120420 in different classification were shown with minimums, the 25 th percentiles, medians, the 75 th percentiles, and maximums. The significance level (α) was adjusted to 0.016 (0.05/3) for multiple pairwise comparisons according to the Mann-Whitney test after overall comparisons of multiple groups according to the Kruskal-Wallis test, * P < 0:05 and * * P < 0:01.

Oxidative Medicine and Cellular Longevity
We found that NR_120420 showed a potential value in predicting the "TACI" AIS subtype; the sensitivity and the specificity reached 85.7% and 84.6%, respectively. Cerebral infarction volume of the patients with TACI is larger than that of the patients with PACI and POCI. More lncRNAs were released from injured brain cells and entered the blood flow via the disrupted blood-brain barrier in these patients. As a result, lncRNA in the peripheral whole blood of them would be significantly different from the CTRL group. The research of our group showed that NR_120420 knockdown inhibited apoptosis after cerebral infarction by downregulating the phosphorylation of a subunit of NF-κB (P65) [18].
Recently, Wang et al. have also studied the role of another circulating lncRNA H19 in predicting AIS; they examined an AIS patient whose onset-to-admission time was less than 3 hours and reported that lncRNA H19 in plasma, lymphocytes, and neutrophils had a sensitivity and specificity of 0.806 and 0.920, 0.556 and 0.920, and 0.75 and 0.72 in predicting AIS, respectively [19]. Our result is comparable to theirs; however, we used the peripheral whole blood samples rather than different blood component, which may be faster in clinic. In addition, the included patients were different; our study included patients whose onset time was less than 6 hours while the aforementioned study included patients whose onset time was less than 3 hours. A recent study showed that the expression profile of whole blood lncRNAs after ischemic stroke changed significantly with time [16].
A limitation of our study was the small sample size for microarray analysis. In addition, this lncRNA microarray technology lacks the ability to unravel novel features of splice isoforms or fusion transcripts compared to RNA-seq technology. Besides, we will carry out investigation about the peripheral whole blood expression of this lncRNA in hemorrhagic patients in the future in order to clarify the exclusive role of this lncRNA for cerebral infarction and make our conclusion be more reliable and specific.

Conclusion
Our study indicated that NR_120420 could predict the total anterior circulation infarction with high sensitivity and specificity and hence could be potentially used as a biomarker for total anterior circulation infarction in AIS patients.

AIS:
Acute ischemic stroke qPCR: Quantitative polymerase chain reaction OCSP: Oxford Community Stroke Project CTRL: Control ROC: Receiver operating characteristic TACI: Total anterior circulation infarction lncRNAs: Long noncoding RNAs ncRNAs: Noncoding RNAs PAC: Partial anterior circulation infarction POCI: Posterior circulation infarction AUC: Area under the curve Ct: Cycle threshold ANOVA: Analysis of variance SD: Standard deviation DM: Diabetes mellitus CHD: Coronary heart disease SBP: Systolic blood pressure DBP: Diastolic blood pressure TC: Total cholesterol TG: Triglyceride LDL: Low-density lipoprotein HDL: High-density lipoprotein.

Ethical Approval
The study was approved by the Medical Ethics Committee of Shanghai Changhai Hospital, and all the experiment procedures were carried out according to the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

Consent
Written informed consent was obtained after all candidates were informed of possible research risks.

Disclosure
The funders had no role in the study design; the collection, analysis, and interpretation of data; the writing of the report; and the decision to submit the article for publication. This article has not used any copyrighted material from other sources (including the Internet).