The protective role of microRNA (miR)-135b in cerebral neurons has been previously identified. However, to the best of our knowledge, the involvement of miR-135b in acute ischemic stroke has yet to be elucidated. The present study aimed to investigate the expression profile of miR-135b in peripheral blood obtained from patients with acute ischemic stroke. A total of 76 patients with acute ischemic stroke were selected as the case group, which included 33 cases of aorta atheromatous plague, 19 cases of cardioembolism, 16 cases of small arterial occlusion and 8 cases with unknown causes. In addition, 60 healthy subjects were selected as the control group. Reverse transcription-quantitative PCR was used to measure the expression of miR-135b in the peripheral blood of the patients. The National Institutes of Health Stroke Scale (NIHSS) score was used to evaluate the severity of acute ischemic stroke. The relationship between miR-135b levels and acute stroke was subsequently analyzed. The expression of miR-135b in the peripheral blood of the case group was found to be significantly higher compared with that in the control group. By contrast, the expression levels of miR-135b in the case group did not differ significantly between the different etiology types of acute ischemic stroke. In addition, a significant positive correlation was observed between levels of miR-135b expression and NIHSS scores. Further analysis demonstrated that hypertension, hyperglycemia, platelet count, international normalized ratio and miR-135b were risk factors for acute ischemic stroke. Based on bioinformatics analysis, a conserved binding site for miR-135b was identified in the 3'-untranslated region of the transient receptor potential cation channel subfamily C member 6 (TRPC6). Dual luciferase reporter and western blot analysis showed that TRPC6 was a target gene of miR-135b. In conclusion, data from the present study suggest that elevated expression of miR-135b in the peripheral blood of patients with acute ischemic stroke is closely associated with disease severity.
Stroke, which is also known as cerebral infarction, is the sudden deprivation of blood supply in the brain (
In recent years, studies have demonstrated that microRNAs (miRs/miRNAs) serve important roles in a number of diseases, including vascular disease, metabolic disease and cancer (
The neuroprotective role of miR-135b in SH-SY5Y cells and stress-enhanced memory has been previously demonstrated (
Therefore, the present study aimed to measure the expression of miR-135b in the peripheral blood of patients with AIS and to analyze the relationship between miR-135b expression and disease etiology and severity to assess the occurrence of miR-135b in AIS.
A total of 76 patients with AIS who were treated at Southwest Hospital (Chongqing, China) between January 2018 and April 2018 were selected as the case group (IS group) based on AIS guidelines (
In the IS group, 5 ml peripheral blood was collected on the day of admission and 14 days after admission, whilst 5 ml peripheral blood was obtained from the control group (NC group) on the day of physical examination. Subsequently, the serum samples were kept at -80˚C until further use. Sex, age, blood glucose, blood pressure and total biochemical indicators, including total cholesterol, glycerol, platelet count and blood zymogen time/international normalized ratio (INR), were routinely recorded at 8:00 a.m. every day for 14 days. The severity of disease affliction was evaluated according to the National Institutes of Health Stroke Scale (NIHSS) (
Extraction of total RNA from serum was performed via enrichment using an RNeasy Micro kit (cat. no. 74004; Qiagen GmbH) according to the kit instructions. RNA concentration and purity were determined using a BioPhotometer™ Plus nucleic acid protein analyzer (Eppendorf) where samples with a total RNA absorbance of 1.8-2.0 were used for subsequent experiments. A total of 10 µl RNA was used to assess RNA quality by denaturing agarose gel electrophoresis. RNA samples were stored at -80˚C.
The RT reaction for mature miR-135b was performed using a PrimeScript RT reagent kit (Takara Bio, Inc.) based on the manufacturer's protocols. RT products were stored at -20˚C. The subsequent qPCR mix was made up to 20 µl with the following: fluorescent quantitative probe reaction mixture (10 µl) (2X ProbeMixture), 1 µl template cDNA, 1 µl 20X miRNA probe, 1 µl amplification primer premix and 7 µl RNase-free ddH2O. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 10 min, followed by 45 cycles of 95˚C for 15 sec and 60˚C for 1 min. U6 was used as external reference for the standardization of extraction and reverse transcription. Relative mRNA expression was normalized to U6 using the 2-∆∆Cq method (
PC12 cells were purchased from American Type Culture Collection and cultured as described previously (
miR-135b mimic (5'-UAUGGC UUUUCAUUCCUAUGUGA-3'), mimic control (5'-UAU AUCG UGUUAUUAGCGUUCCU-3') (both from Shanghai GenePharma Co., Ltd.), inhibitor (5'-UCACAUAGG AAUGAAAAGCCAUA-3') or inhibitor control (5'-UUCAUC GUGUUAUUAGCGUUCCU-3') was transfected into PC12 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. In brief, PC12 cells were cultured at a density of 1x106 cells/well and miR-135 mimic, mimic control, inhibitor or inhibitor control were transfected after 24 h at a final concentration of 20 nM. At 48 h after cell transfection, transfection efficiency was detected using RT-qPCR.
TargetScan (
PC12 cells were collected and treated with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1% (v/v) phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), 0.3% (v/v) protease inhibitor (Sigma-Aldrich; Merck KGaA) and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma-Aldrich; Merck KGaA). Western blots were performed as previously described (
Data were represented as the mean ± SD. Statistical analysis was performed on SPSS 20.0 (IBM Corp.). Two-tailed unpaired Student's t-test was used for comparisons between two groups. One-way ANOVA multiple comparison test followed by Tukey's post hoc test was used for comparisons between >2 groups. Chi square test was performed for comparing categorical variables of the clinical data. Receiver operating characteristic (ROC) curves was used to assess miR-135b as a diagnostic biomarker, where the area under the curve (AUC) was calculated. Correlation between miR-135b expression and NIHSS scores was analyzed using the Pearson correlation coefficient (r) method; logistic regression analysis was used to explore the risk factors affecting AIS. Kaplan-Meier analysis followed by log-rank test was used to analyze the overall survival rate based the levels of miR-135b in peripheral blood. P<0.05 was considered to indicate a statistically significant difference.
No significant differences in sex, mean age, smoking, alcohol consumption, total cholesterol and triglyceride levels were found between the IS and control groups (
RT-qPCR was used to measure the expression levels of miR-135b in the peripheral blood of IS and control groups. Serum miR-135b expression levels was revealed to be significantly higher in the IS group (4.20±2.35) compared with those in the NC group (1.00±0.83) (
No significant differences were found in the expression levels of miR-135b among the 33 cases of aortic atheromatous plague, 19 cases of cardioembolism, 16 cases of small arterial occlusion and 8 cases with unknown causes (
A significant positive correlation was found between miR-135b expression and the NIHSS scores in the peripheral blood of patients in the IS group (r=-0.835; P<0.001;
Logistic multivariate regression analysis was performed using AIS as the dependent variable, whilst hypertension, hyperglycemia, platelet count, INR and miR-135b expression were used as independent variables. Hypertension, hyperglycemia, platelet count, INR and miR-135b expression were found to be risk factors for AIS, as shown in
The sensitivity and specificity of peripheral blood miR-135b in the diagnosis of IS were analyzed using ROC curve analysis. The AUC of miR-135b was 0.78 (95% CI, 0.69-0.87;
Association between serum miR-135b levels and overall survival rates of patients with AIS was next evaluated. The median value of 2.14 was applied for miR-135b expression in all 76 patients with AIS for separating those with high miR-135b expression (n=27) from low miR-135b expression (n=49). Kaplan-Meier curves indicated that patients with high miR-135b expression exhibited significantly poorer survival compared with those with low miR-135b expression (P=0.032;
Based on the aforementioned observations, the potential target genes of miR-135b was subsequently analyzed. Analysis using the TargetScan software identified a conserved binding site in the 3'-UTR of TRPC6 (
At present, clinicians place heavy reliance on the presentation of clinical symptoms and signs from patients combined with head imaging to diagnose AIS (
miRNAs are single-stranded RNA molecules 18-25 nucleotides in length that regulate gene expression in many cellular processes (
The diagnostic value of miR-135 in distinguishing between patients with AIS and healthy controls was subsequently evaluated. The present data showed that the sensitivity and specificity of miR-135b for IS diagnosis revealed an AUC of 0.78 (95% CI, 0.69-0.87). When the cut-off value was set to 40 fmol/l, the diagnostic sensitivity was 79.25% and the specificity was 64.71%. These findings suggested that serum miR-135b exhibited a certain accuracy and feasibility as a biomarker of AIS. However, a gap remains between the high specificity and high sensitivity of this marker. Therefore, the combined detection of miRNAs with other protein markers associated with ischemic stroke should be examined in future research.
A number of reports have previously demonstrated that circulating serum miRNAs may serve as potential non-invasive biomarkers for patients with AIS (
The possible target genes of miR-135b were next investigated. TRPC6, an important regulator of the neuronal survival pathway (
It should be noted a number of limitations remain associated with the present study. A time profile of miR-135b would be more practical, which will assist in elucidating the role of miR-135b during disease onset and AIS prognosis. However, due to the study design, the number of blood samples obtained remain insufficient. In future studies, a larger cohort of samples should be collected, where miR-135b expression will be studied over a period of time. In addition, the present study mainly focused on measuring the expression of miR-135b in the serum of patients with AIS and to analyze the relationship between its expression levels and the etiology and severity of the disease, thereby investigating miR-135b occurrence in AIS. Investigations into the clinical values of miR-135b in differentiating AIS, hemorrhagic stroke and other brain diseases remain an attractive line of future research. Specifically, samples obtained from patients with other brain diseases could be investigated to elucidate the diagnostic value of serum miR-135b levels further.
In conclusion, miR-135b was found to be closely associated with the severity of patients with AIS in the present study, where high levels of miR-135b in the peripheral blood can be used to indicate the severity of neurological damage and are risk factors for AIS. These findings may contribute to the future clinical diagnosis and prognosis of AIS, however, the importance of miR-135b intervention in the treatment of AIS warrants further investigation.
Not applicable.
The present study was supported by a grant from the Southwest Hospital, Third Military Medical University (grant no. TMMUSH-20150935).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
SY performed the experiments, analyzed the data, wrote the manuscript, designed the experiments, analyzed the data and gave final approval of the version to be published. XYZ, MH, JJW and XMQ performed part of the RT-qPCR experiments. All authors read and approved the final version of the manuscript.
The present study was approved by the Research Ethics Committee of Southwest Hospital, Third Military Medical University (Chongqing, China). All patients provided written informed consent.
Not applicable.
The authors declare that they have no competing interests.
Comparison of miR-135b expression between IS and healthy control group. Reverse transcription-quantitative PCR was performed to analyze the levels of miR-135b in patients with IS and healthy controls. ***P<0.001. miR-135b, microRNA-135b; IS, ischemic stroke; NC, control.
Comparison of miR-135b expression among the four etiological groups of IS with the IS group. No significant difference was observed in expression levels of miR-135b among the aorta atheromatous plague, cardioembolism, small artery occlusion and unknown causes groups. IS, ischemic stroke; miR-135b, microRNA-135b.
Correlation analysis between peripheral blood miR135b expression and NIHSS scores. Pearson correlation analysis showed that miR-135b correlated positively with NIHSS scores in the peripheral blood of patients in the IS group. IS, ischemic stroke; miR-135b, microRNA-135b; NIHSS, National Institutes of Health Stroke Scale.
Analysis of diagnostic ability of using miR-135b expression in the peripheral blood for IS. Receiver operating characteristic analysis showed that peripheral blood microRNA-135b could differentiate patients with IS from healthy controls. IS, ischemic stroke.
Comparison of overall survival rates between patients with high miR-135b expression and low miR-135b expression. Kaplan-Meier curve analysis suggested that patients with low levels of miR-135b exhibited significantly poorer survival compared with patients with high levels of miR-135b. miR-135b, microRNA-135b.
TRPC6 is a target gene of miR-135b. (A) TargetScan analysis identified a conserved binding site for miR-135b in the 3'-UTR of TRPC6. (B) Dual-luciferase assay showed that co-transfection with miR-135b significantly reduced the relative luciferase activity of pmirGLO-TRPC6-3'UTR but not pmirGLO. (C) Transfection with the miR-135b mimic significantly increased the levels of miR-135b and (D) significantly reduced the expression of TRPC6 in PC12 cells compare with the NCm group. (E) Transfection with miR-135 inhibitor significantly reduced the relative expression of miR-135b and (F) significantly increased the expression of TRPC6. *P<0.05; ***P<0.001. 3'UTR; 3'-untranslated region; NC, negative control transfected with negative control mimics/inhibitor; miR-135b, microRNA-135b; TRPC6, transient receptor potential cation channel subfamily C member 6; RLU, relative luminometer unit; NCm, NC mimic; NCi, NC inhibitor.
General clinical data for patients with AIS and healthy controls.
Indicator | Healthy controls (n=60) | AIS patients (n=76) | P-value |
---|---|---|---|
Sex (male/female) | 41/19 | 55/21 | 0.621 |
Average age (years) | 53.72±11.26 | 54.68±10.98 | 0.815 |
Smoking [n (%)] | 27 (45.00) | 39 (51.35) | 0.464 |
Drinking [n (%)] | 21 (35.00) | 33 (43.42) | 0.319 |
Systolic blood pressure (mmHg) | 121.15±10.28 | 152.62±10.75 | <0.001 |
Diastolic blood pressure (mmHg) | 79.84±8.63 | 98.52±6.85 | <0.001 |
Previous cerebrovascular event [n (%)] | 0 | 25 (32.9) | <0.001 |
Family cerebrovascular event [n (%)] | 0 (0.0) | 18 (23.7) | <0.001 |
Homocysteine level (μmol/l) | 11.46±1.85 | 22.56±6.35 | 0.043 |
Uric acid (μmol/l) | 235.56±73.11 | 359.03±96.71 | 0.031 |
FBG (mol/l) | 5.38±1.32 | 7.25±1.96 | <0.001 |
TC (mmol/l) | 5.12±1.38 | 5.24±1.76 | 0.589 |
TG (mmol/l) | 1.46±0.35 | 1.44±0.48 | 0.762 |
Platelet count (x109/l3) | 215.82±33.56 | 175.78±26.84 | <0.001 |
INR | 1.12±0.26 | 2.63±0.72 | <0.001 |
NIHSS score | N/A | 18.82±5.14 | N/A |
Intravenous thrombolytic therapy [n (%)] | 0 (0.0) | 26 (34.2) | N/A |
Data were represented as the mean ± SD unless otherwise stated. AIS, acute ischemic stroke; FBG, fasting blood glucose; TC, total cholesterol; TG, triglyceride; INR, international normalized ratio; NIHSS, National Institutes of Health Stroke Scale.
Multivariate logistic regression analysis results.
Indicators | HR | 95% CI | P-value |
---|---|---|---|
Hypertension | 2.756 | 1.902-4.321 | <0.001 |
Hyperglycemia | 1.862 | 1.543-2.356 | 0.014 |
Platelet count | 2.094 | 1.678-2.635 | 0.001 |
INR | 1.589 | 1.163-2.253 | 0.025 |
miR-135b expression | 2.356 | 1.825-3.287 | 0.002 |
INR, international normalized ratio; miR-135b, microRNA-135b; HR, hazard ratio, CI, confidence interval.