The lower expression of circulating miR‐210 and elevated serum levels of HIF‐1α in ischemic stroke; Possible markers for diagnosis and disease prediction

Abstract Background Stroke, either due to ischemia or hemorrhage, causes acute neurological damages to the brain. There is shortage of reliable biomarkers for ischemic stroke (IS), and we therefore investigated the serum concentrations of microRNA‐210 (miR‐210) and hypoxia inducible factor‐1α (HIF‐1α), as possible diagnostic and/or prognostic markers for IS. Methods Serum samples were acquired from 52 IS patients and their healthy counterparts at five time points: upon admission, 24 and 48 h after admission, upon discharge and 3 months later. Serum levels of miR‐210 and HIF‐1α were respectively analyzed using real time RT‐PCR and ELISA. Diagnostic and prognostic accuracy tests were performed to assess the value of suggested biomarkers. Results IS patients demonstrated higher levels of serum HIF‐1α and lower miR‐210 in comparison to the healthy subjects. MiR‐210 was suggested to be a weak diagnostic biomarker at the time of admission (AUC = 0.61; p = 0.05), while HIF‐1α was an acceptable diagnostic marker for IS (AUC = 0.73; p < 0.0001). The higher expression of miR‐210 and lower levels of HIF‐1α were associated with better survivals in IS patients. Conclusions Serum miR‐210 is a weak diagnostic marker of IS. Serum HIF‐1α is a better biomarker in diagnosing IS patients but further work in larger groups, including those with hemorrhagic stroke is necessary to confirm its diagnostic utility. Similarly, the prognostic potentiality of miR‐210 and HIF‐1α was acceptable but needs bigger sample size and longer follow‐up to be statistically confirmed.

cerebrospinal fluid), or methods that require expensive technology such as positron emission tomography (PET). 5 There remains a lack of established, sensitive, and specific non-invasive methods such as serum biomarkers for the diagnosis and predicting the outcome of IS.
Noncoding RNAs (ncRNAs) play significant roles in controlling gene expression and physiologic cellular functions. 6 These ncRNAs are highly expressed in mammalian brains and their alterations appear to have a remarkable role in brain ischemia and post-stroke improvement. 7 Recent reports have shown that a small, endogenous class of ncRNAs known as microRNA (17)(18)(19)(20)(21)(22)(23)(24)(25) nucleotides; miRNA) 8 can modulate the transcription or translation of genes and thus regulate the expression of mRNAs and proteins. 9,10 MiRNAs are extensively stable in plasma, serum, urine, and CSF and could be introduced as diagnostic, prognostic, or remedial biomarkers for many diseases. 4,11 One of the most important groups of miRNAs, upregulated by hypoxia, is called 'Hypoxamirs'. 12 Hypoxia, the situation of inadequate oxygen nourishment to tissues, results from a reduction in oxygen availability, insufficient oxygen transport, or disability of tissues to utilize oxygen. Hypoxia is crucially involved in both acute and chronic ischemia. MiRNA-210 (miR-210) (MI0000286) is one the most predominant hypoxiamirs, 13 activated by hypoxia inducible factor-1 (HIF-1). 14 It has been proposed that miR-210 could be a novel circulating biomarker for the diagnosis and prognosis of IS. 15 Studies have also indicated that miR-210 is a crucial determinant in adjusting several hypoxia-associated pathways in physiological and pathological conditions. 16 MiR-210 can prevent apoptosis by targeting multiple transcripts involved in many sides of cellular responses to hypoxia, protect stem-cell survivance, terminate mitochondrial metabolism, promote glycolysis, and induce angiogenesis. 17 Enhanced HIF-1 expression, a major characteristic of response to hypoxia, is a member of hypoxia inducible factors (HIFs). In hypoxia, the alpha subunit of HIF-1 (HIF-1α) is stabilized and upregulated. 18 Targetscan information has predicted that miR-210 could be the target of HIF-1α (http://www.targe tscan.org/vert_72/) and its expression could be regulated by hypoxia inducible transcription factors upon exposure to low oxygen. 14,19 The aim of this study was to examine the alterations of miR-210 and HIF-1α in patients with IS, assessing their diagnostic and prognostic values and monitoring their expressional changes in a 3 months follow-up.

| Study subjects and Blood sample collection
We enrolled fifty-two IS patients, consecutively, after following the confirmation of the diagnosis by the neurologist at Sayyad Patients with a history of cancer, hematologic disorders, renal or liver failure, or recurrent stroke were not included in the study.
Serum samples were also obtained from 52 age-and sex-matched volunteers. The latter were healthy controls, who attended the Deziani Specialized and Sub-Specialized Clinic in Gorgan, Iran for a routine health check-up. Individuals with cardiovascular disorders, history of stroke, or kidney or liver failure, and diabetes were excluded. Written informed consent was obtained for all participants, in accordance with the declaration of Helsinki [20].
The demographic and laboratory data of all participants are reported in our previous research work [21]. The outcome of the disease was followed for 3 months after initial IS attack by the modified Rankin Scale (mRS). The mRS is a measure that evaluates disability in patients with stroke to check their recovery or continued disability over time. A score of 0 indicates no disorder, while 5 demonstrates a condition that indicates that care is constantly required, and a patient with 6 score is deceased. 21 The mRS scores were considered as favorable (0-2) and unfavorable (3)(4)(5)(6) outcomes. National Institute of Health stroke scale (NIHSS) scores were also used for the quantification of neurological deficit. Blood samples were collected in a five-step procedure; at the time of admission, and then after 24 and 48 h after the stroke event, at the time of discharge, and 3 months later. Samples were centrifuged to remove blood cells, and the serum was immediately divided into two aliquots. One aliquot was immediately processed for the total RNA extraction and the other one was stored at −80°C until use.

| HIF-1α ELISA assay
Serum HIF-1α was measured using a commercial ELISA kit (Elabscience) following the manufacturer's protocol. The limit of detection (LOD) for the utilized ELISA kit was 62.50-4000 pg/ml, while the sensitivity was 37.50 pg/ml, and both intra-and inter-CV (coefficient of HIF-1α) were less the 10%, as disclosed. All samples were analyzed in duplicates and the results were reported as nanograms (ng) of HIF-1α per mL. Linear regression was used to quantify the absorbance of 450 nm against the reference absorbance of 630 nm using a Chromate 4300 microplate reader (Biotek).

| Statistical analyses
The study sample size was calculated based on research by C.S.
Gan et al. 24 The power of the test was also calculated to be 90%.
The Kolmogorov-Smirnov test for goodness of fit was utilized to test the normality in this study. In order to evaluate the diagnostic utility of each variable, ROC curve analyses were conducted.
Pearson and Spearman correlation studies were applied to correlate quantitative variables. Logistic regression was performed to demonstrate the combined ROC curve analysis and predict the performance of miR-210 + HIF-1α. Linear regression was used to model the relationships between a scalar response (dependent variable) and one or more explanatory variables (independent variables). Performing the comparisons between the means of more than two groups, we used Two-way ANOVA or Kruskal-Wallis and

| The serum concentration of miR-210 and HIF-1α
Serum miR-210 in the IS patients was lower than the normal controls, at the time of admission (p = 0.0003) ( Figure 1A). Serum HIF-1α was higher at the time of admission in patients compared with the normal F I G U R E 1 The serum steady-state concentration of miR-210 and HIF-1α levels; miR-210 was down-regulated in IS patients (A), while HIF-1α was expressed in higher quantities (B). The calculations of the expression levels for miR-210 were conducted by 2 −ΔCt method. To compare the means between the two groups, students' T-test was used (Patients: 52, Healthy subjects: 52). Statistics on each scattered plot demonstrates Mean ± SD. The level of significant p-values was 0.05 controls (p < 0.0001) ( Figure 1B). Serum levels of miR-210 were significantly different between the time of admission and 3 months after the stroke incidence (p = 0.0111) (Figure 2A), but serum HIF-1α did not markedly change at different time points ( Figure 2B).

| Diagnostic performance of miR-210 and HIF-1α
The

| The prognostic value of miR-210 and HIF-1α
According to the outcome of the disease after 3 months of followup, patients were divided into 2 groups; those who died (mean age = 73.5 years) and those who survived (mean age = 62.8). Based on the serum concentrations of both potential biomarkers, subjects were divided into categories of high and low levels according to the optimum cut-off points derived from ROC curve analyses. Log-rank

| Correlation analyses
According to the mRS scores, patients were divided into 5 groups.

| DISCUSS ION
Ischemic stroke is caused by the occlusion of one or more cerebral arteries, leading to hypoxia, 25,26 and resulting in a diminution of oxygen supply to the brain. 7 Circulating microRNAs and diseasesassociated blood proteins may be helpful in discriminating patients with and without disease, and predicting outcomes. 27 MiR-210 is a Hypoxamir that is activated by hypoxia inducible factor-alpha (HIF-1α). 15,25 Given the role of hypoxia in stroke pathogenesis, the investigation of serum miR-210 and HIF-1α as biomarkers were addressed in our present study.
MiR-210 was significantly lower in stroke patients compared to the control group and we also found that there was a significant difference between the mean of miR-210 at the time of admission and 3 months after stroke. Several previous studies expressed that the lower expression of miR-210 could increase PGE2 expression, in association with HIF-1α higher levels. 28 The lower expression of miR-210 may be associated with the deactivation of PI3K/Akt pathway which mediates neuroprotective effects and the arrested signaling could promote unrestricted stroke. 29 Moreover, miR-210 downregulation is associated with the elevated expression of p53 tumor suppressor gene within DNA repair process, which is involved in the modification of stroke severity. 30 Serum HIF-1α levels were higher in stroke patients than controls.
However, HIF-1α protein expression did not change significantly at different time points. The serum levels of HIF-1α, as the major modulator of pathophysiologic responses to hypoxia, are mainly controlled by being produced and degeneration in a cycle. The higher F I G U R E 4 Survival analyses; IS patients were subdivided into two categories of high and low expression on the basis of the optimum cut-off points of suggested biomarkers. Log-rank test results showed that higher expression of miR-210 was able to predict the outcome (survival) of the disease after 90 days of follow-up (A) also, lower expression of HIF-1α was capable of predicting the outcome of IS (B) levels of HIF-1α in response to hypoxia-associated processes are controlled by an oxygen-related pathway known as Hippel Lindau protein (pVHL). 31 This pathway is probably deactivated during the first stages of ischemic stroke, in which the oxygen levels decline to a critical level. 32 However, HIF-1α is believed to be an early acute phase marker of IS 33 and by increasing the oxygen level, the pVHL pathway activates and consequently, the HIF-1α serum levels fall into a consistent level. 34 Moreover, several studies have reported that healthy individuals may express ample levels of HIF-1α, in the absence of specific clinical diseases. 35 This may partially explain the lack of significant difference between HIF-1α levels at different time points, while the small sample size in our study and regular methodological errors of ELISA assay still remain major limitations that should be considered.
According to the ROC curve analyses and calculated AUC, miR-210 could be introduced as diagnostic marker (however, with poor performance), while serum HIF-1α might be a helpful marker with acceptable sensitivity and specificity. Although the results of Kaplan-Meier analyses were not significant, the higher expression of miR-210 and lower levels of HIF-1α were associated with better survival in IS patients. Moreover, serum miR-210 and HIF-1α had no significant relationship with NIHSS, nor with mRS at 3 months after stroke and were inversely related with each other at the time of admission. As Consistent with our findings, there have been several previous reports that miR-210 is reduced in the serum of stroke patients compared to controls. 15,30,36,37 Other studies have also identified that miR-210 was a powerful diagnostic marker. 15,30 Although our findings support that miR-210 could be introduced as a diagnostic marker for IS, it is not of great value and categorized as a poor biomarker. Other investigators found that the expression of this marker in patients with mRS<2 was higher than in those with mRS>2, 36,38 while we found no significant difference between these groups.
In accordance with our findings, previous studies have shown that serum HIF-1α concentrations were higher in stroke patients than in the control groups. 18,39,40 These investigators found that concentrations of serum HIF-1α were significantly higher in individuals with mRS>2 than in individuals with mRS≤2, but no correlation between NIHSS and HIF-1α levels. 39 Based on our study that HIF-1α could be introduced as a fair diagnostic marker in addition to other diagnostic tools with less invasion. Moreover, a weak correlation was found between miR-210 and HIF-1α at the time of admission, which could delineate the regulatory effects of miR-210 on the expression of HIF-1α. The lack of association between miR-210 and HIF-1α after 3 months of follow-up, could be due to the rather small sample size of our study. However, these findings are suggested to be evaluated in further investigations with bigger sample size. Since the severity of the disease was different among patients upon admission and during the study, the treatment approach was also different and it was not possible to perform subgroup analysis regarding the treatment strategies. Although our research work was associated with F I G U R E 5 Alterations of HIF-1α and miR-210 after 3 months of follow-up in different categories of mRS; miR-210 was downregulated in IS patients of the MRS 5 category (A), while no remarkable change was observed in the expression of HIF-1α between 5 groups of MRS (B). IS patients falling in the 6 th category of mRS had passed away and no record of HIF-1α or miR-602 was available. One-Way ANOVA and Tukey's post-test or Kruskal-Wallis and Dunn-Bonferroni post-test were used to evaluate the differences between the means of various groups. Bar charts show Mean ± SD for each value. Level of significant p-values was 0.05 the limitations which are mostly affected by the small sample size, miR-210 and HIF-1α showed acceptable performance in predicting the fate of the disease in IS, which should be confirmed with more sophisticated further studies. It is strongly suggested to perform future research on larger sample sizes, recruiting different subgroups of ischemic stroke as control groups (at various stages, with diverse outcomes and different therapeutic approaches), and analyzing the prognosis in a longer period of time. Moreover, these suggested markers could be evaluated on other ischemic disorders to be possibly introduced as general biomarkers for ischemia.

| CON CLUS ION
We found that the serum levels of miR-210 were lower, while serum HIF-1α levels were higher in stroke patients. MiR-210 was introduced as a poor biomarker for IS, while HIF-1α was categorized as a fair marker in the diagnosis of IS. Moreover, both miR-210 and HIF-1α showed acceptable performance in predicting the fate IS patients. However, more sophisticated research works with a bigger sample size would be necessary to evaluate the diagnostic/ prognostic value of these markers in distinguishing IS patients from hemorrhagic stroke. According to the significant role of Hypoxamirs including miR-210 and targeted molecules such as HIF-1α in ischemic disorders, our study showed that HIF-1α and miR-210 could be suitable diagnostic and prognostic markers for IS.

ACK N OWLED G M ENTS
All data in this article was obtained from an M. Sc. thesis in the field of Clinical Biochemistry written by Mina Rahmati at Gorgan School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.

CO N FLI C T O F I NTE R E S T S
None.

E TH I C A L A PPROVA L
The study was approved by the Committee of Ethics, Golestan

DATA AVA I L A B I L I T Y S TAT E M E N T
Data supporting the findings of this study are available from the corresponding author upon reasonable request.