Acute cerebral infarction (ACI) is a series of neurological dysfunction symptoms caused by ischemia and hypoxia of local brain tissue due to insufficient blood supply.1 Research shows that the death/disability rate of ACI within 1 year is high, which is seriously harmful to people's health.2 The diagnosis and treatment mainly focus on early detection, treatment and prevention.3,4 There have been studies on the diagnostic value of C‐reactive protein and interleukin‐6 in ACI, but their specificity is not high.3–5 At present, the diagnosis of ACI depends on the Stroke Scale score, computed tomography (CT), magnetic resonance imaging (MRI) and other imaging methods.6 Imaging examination is of great significance to exclude hemorrhagic stroke, but CT examination cannot show the ischemic focus well in the early stage of infarction, while MRI examination is not applicable for some patients with metal stents.4,6 Therefore, it is important to find sensitive biomarkers for accurate diagnosis and prognosis evaluation of ACI.
MicroRNAs (miRs) are endogenous small noncoding RNAs involved in gene expression regulation.7,8 As intracellular RNAs, they negatively regulate gene expression at the posttranscriptional level by binding to the 3′ untranslated end of messenger RNA (mRNA) and have become a hot topic because of their potential as therapeutic targets.9 Previous studies have shown that microRNAs are abundantly present in body fluids in a very stable form, including serum, plasma and urine without cells, and can reflect the pathophysiological state of the body through their expression in body fluids.10,11 An increasing number of studies have proven the potential value of microRNAs as a new biomarker in the diagnosis of ACI.12,13 However, we still lack knowledge about miRs in the progression of ACI.
In the present study, we mainly focused on miR‐409‐3p, which was found to be dysregulated in several neurodevelopmental disorders.14,15 For the first time, we showed novel data that miR‐409‐3p was significantly upregulated in the serum of patients with ACI, which may shed light on the diagnosis and therapy of ACI in the clinic.
From January 2017 to March 2018, 80 inpatients within 9 h of ACI onset (5.4 ± 3.2 h, 2.1 ~ 8.6 h) were collected, including 53 males and 27 females, with an average age of 57.0 ± 21.1 years (38–79 years). All patients had not been treated with dilatation, vasodilation, and thrombolysis before blood collection, and patients with trauma or operation, as well as other cancer, infection or immune diseases were excluded. Thirty healthy people in the same period were selected as the control group, including 20 males and 10 females, with an average age of 56.0 ± 20.9 years. There was no significant difference in sex, age, hypertension, diabetes or hyperlipidemia between the two groups (P > .05).
According to the National Institutes of Health Stroke scale (NIHSS),16 patients with AIS were divided into three groups: mild group (NIHSS <4), moderate group (4 ≤ NIHSS ≤15) and severe group (NIHSS >15).
Based on the results of MRI imaging examination of patients with AIS, the infarct volume was evaluated according to the Pullicino method17 and divided into the small infarction group (infarct volume < 5 cm3), middle infarction group (5 cm3 ≤ infarct volume < 10 cm3) and large infarction group (infarct volume ≥ 10 cm3).
This study was approved by the ethics committee of Xuzhou Central Hospital, and informed consent was obtained from the patients. Details for the patients are shown in Table 1.
TABLEBaseline clinical characteristics of the patients with ACI and normal controls| Parameter | Patients with ACI (n = 80) | Healthy controls (n = 30) | P value |
| Mean age | 57.0 ± 21.1 | 56.0 ± 20.9 | 0.763 |
| Sex (M/F) | 53/27 | 20/10 | 0.658 |
| Stroke risk factors | |||
| Hypertension | 48 (60%) | 15 (%) | 0.562 |
| Diabetes mellitus | 42 (52.5%) | 5 (16.7%) | 0.032 |
| Atrial fibrillation | 20 (25%) | 7 (23.3%) | 0.827 |
| Hyperlipidemia | 48 (60%) | 4 (13.3%) | 0.016 |
| NIHSS score | 7.89 ± 1.42 | NA | NA |
Five milliliters of peripheral blood was taken from patients with ACI within 9 h of stroke onset and from the control group. Then, the blood was centrifuged at 1600 × g for 10 min. After that, the serum was transferred into a new tube and centrifuged at 20,000 × g at 4°C for 15 min. The supernatant was then collected and kept at −80°C until use.
The total volume of reverse transcription was 25 μl, including 1 μl (250 ng) of RNA, 1 μl of primer, 2 μl of dNTP, 2.5 μl of 10 × sample buffer, 0.3 μl of RNA inhibitor (NEB), 0.5 μl of MMLV (NEB), and 17.7 μl of RNA‐free ddH2O. Reverse transcription conditions were as follows: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min and 4°C for preservation. The total volume of fluorescent quantitative PCR was 20 μl, including 2 μl of cDNA, 10 μl of 2 × SYBR Green (Takara), 1 μl of upstream and downstream primers, and 6 μl of sterile ddH2O. The reaction conditions were as follows: 95°C for 2 min; 35 cycles of 95°C for 30 sec and 60°C for 1 min; 72°C for 5 min; and 4°C for preservation. The fluorescence intensity of PCR was obtained by instrument, and the expression of genes was calculated using the 2‐∆∆Cq method.18
The levels of CTRP3 (cat no. 580200–1, Amyjet Scientific Co., China) in the serum were quantified using ELISA assays following the manufacturer's protocol. In brief, serum samples were homogenized in lysis buffer (50 mmoL/L Tris–HCl, 300 mmoL/L NaCl, 5 mmoL/L EDTA, 1% Triton X‐100 and 0.02% sodium azide) containing a protease inhibitor cocktail (Roche Diagnostics). Lysates were centrifuged at 16,000× g for 15 min at 4°C and levels of CTRP3 (cat no. 580200–1, Amyjet Scientific Co., China) in the supernatants were quantified and were then read at 450 nm using a microplate reader.
PC12 cells were purchased from the Bank of the Chinese Academy of Sciences (China). Cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), streptomycin (100 μg/ml) and penicillin (100 U/ml; Thermo Fisher Scientific, Inc.) in 25‐cm2 culture flasks at 37°C in a humidified atmosphere containing 5% CO2.
First, 6x105 cells were equally seeded in 6‐well plates with 2 ml of DMEM containing serum and antibiotics. miR‐409‐3p mimics, inhibitors or miR negative controls were purchased from GenePharma (Shanghai, China). Simultaneously, miR‐409‐3p mimics, inhibitors or miR negative controls were mixed with HiperFect transfection reagent (Qiagen GmbH, Hilden, Germany) and incubated at room temperature for 10 min. Then, each complex was transfected into two wells containing PC12 cells for 48 h at a final concentration of 10 nM.
TargetScan 7.1 (
The 3′ untranslated region (UTR) of CTRP3, which contains the predicted target site for miR‐409‐3p, was cloned into the pmirGLO luciferase reporter vector (Promega Corporation, Madison, WI, USA). Details of the PCR procedures are described as follows: a hot start step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 55°C for 45 sec and 72°C for 30 sec. Prior to conducting the dual reporter assay, 5 × 104 PC12 cells/well were seeded in 24‐well plates with 500 μl of DMEM and cultured for 18 h. The cells were transfected with the modified firefly luciferase reporter vector (500 ng/μl) mixed with Vigofect transfection reagent (Vigorous, Beijing, China) according to the manufacturer's protocol (see supplemental materials). After continuous exposure of miR‐409‐3p/pmirGLO‐CTRP3 or NC/pmirGLO blank vector for 48 h, the luciferase activities of firefly and Renilla were measured with the Dual‐Luciferase® Reporter Assay system (Promega Corporation) according to the manufacturer's protocol (see supplemental materials).
Cellular protein was extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with protease inhibitor cocktail (1:100, P8340, Sigma‐Aldrich, USA) and was collected following centrifugation at 12,000 × g for 30 min at 4°C. A bicinchoninic protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 15 μg of protein was loaded per lane, separated by 10% SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 8% nonfat dry milk at 4°C overnight. Following three washes with PBS plus Tween 20 (5 min/wash), the membranes were incubated with the primary antibodies at 4°C overnight. Following several washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit and anti‐mouse immunoglobulin G (IgG) or HRP‐conjugated mouse anti‐goat IgG (all 1:5000; Zhongshan Gold Bridge Biological Technology Co., Beijing, China) for 2 h at room temperature and then washed. The blots were then incubated with HRP‐conjugated anti‐IgG secondary antibody (1:5000; OriGene Technologies, Inc., Beijing, China) for 2 h at room temperature and then washed, followed by detection with enhanced chemiluminescent substrate (EMD Millipore, Billerica, MA, USA). GAPDH was used as an internal control. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for density analysis.
To detect cell apoptosis, PC12 cells were washed with 1× PBS three times and analyzed by the Annexin V‐fluorescein (FITC)‐propidium iodide (PI) Apoptosis Kit (Beyotime Institution of Biotechnology, Shanghai, China) according to the manufacturer's protocol (see supplemental materials). Cells were analyzed by an FC500 flow cytometry instrument equipped with CXP software (Beckman Coulter, Bethesda, MA, USA).
Nuclear fragmentation was detected using TUNEL staining with an In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the supplier's instructions. In brief, he transfected cells were fixed using 4% paraformaldehyde for 30 min, followed by incubation with TUNEL buffer for 1 h at 37°C. After rinsing with PBS, the number of TUNEL‐positive apoptotic cells and the total number of cells in five different random high‐power fields were counted using a microscope (Olympus Corporation, Tokyo, Japan) at a magnification of 400. The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL‐positive cells to the total number of cells.
The data are presented as the mean ± standard deviation (SD). Two‐tailed unpaired Student's t‐tests were used for comparisons between two groups. Analysis of variance followed by Tukey's post hoc test was used for multiple group comparisons. Pearson correlation analysis was used to assess the correlation between the Gensini score and plasma miR‐409‐3p levels. A receiver operating characteristic curve (ROC) was used to calculate the area under the receiver operating characteristic curve (AUC) to evaluate the diagnostic value of plasma miR‐409‐3p levels in patients with ACI. All statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, Illinois). P < .05 was considered a statistically significant difference.
First, we evaluated the expression of plasma miR‐409‐3p in patients with ACI. Our data showed that the expression of plasma miR‐409‐3p was significantly higher in patients with ACI than in healthy controls (Figure 1(A)). We then analyzed the level of plasma miR‐409‐3p according to infarct volume. As shown in Figure 1(B), the level of plasma miR‐409‐3p was enhanced significantly in the large infarction group compared with the levels in the middle infarction group and small infarction group (Figure 1(B)). Furthermore, we also evaluated the level of plasma miR‐409‐3p according to the NIHSS score in patients with ACI. Compared with the mild group, a higher plasma miR‐409‐3p level was found in the moderate group, and the highest plasma miR‐409‐3p was identified in the severe group (Figure 1(C)). Meanwhile, a positive correlation was also determined between plasma miR‐409‐3p and NIHSS score (r = 0.865, p < 0.001) (Figure 1(D)). We also evaluated the association between plasma miR‐409‐3p level and other risk factors, such as age and gender. However, no significant correlation was found. Then, we performed receiver operating characteristic (ROC) curve analysis to explore whether miR‐409‐3p could be used as a potential biomarker in the diagnosis of ACI. The area under curve (AUC) of plasma miR‐409‐3p was 0.835 (Figure 1(E)). When the cut‐off value was 4.76, the sensitivity and specificity were 89.5% and 93.8%, respectively. These data indicate plasma miR‐409‐3p may be a useful biomarker in the diagnosis of ACI.
Enlarge this image.1 FIGURE. The expression of miR‐409‐3p was significantly increased in patients with ACI compared with healthy controls. (A) the expression of plasma miR‐409‐3p was higher in patients with ACI than in healthy controls. (B) the level of plasma miR‐409‐3p was enhanced significantly in the large infarction group compared with that of the middle infarction group and the small infarction group. (C) Compared with the mild group, a higher plasma miR‐409‐3p level was found in the moderate group, and the highest plasma miR‐409‐3p level was identified in the severe group. (D) Pearson correlation analysis indicated a positive correlation between plasma miR‐409‐3p and the NIHSS score. (E) ROC analysis was performed to explore the diagnosis value of plasma miR‐409‐3p in ACI patients and healthy controls. *p < 0.05, ***p < 0.001 vs. as indicated
These observations prompted us to explore the possible target genes of miR‐409‐3p. Based on TargetScan, a conserved binding site was found in the 3'UTR of CTRP3 (Figure 2(A)). The dual luciferase reporter assay indicated that miR‐409‐3p significantly suppressed the relative luciferase activity of pmirGLO‐CTRP3‐3'UTR (Figure 2(B)). We evaluated the transfection efficiency, and real‐time PCR analysis demonstrated that transfection of the miR‐409‐3p mimic significantly enhanced the miR‐409‐3p level (Figure 2(C)). Western blot assays indicated that miR‐409‐3p significantly suppressed the expression of CTRP3 in PC12 cells (Figure 2(D)). In contrast, transfection of miR‐409‐3p inhibitor significantly suppressed miR‐409‐3p levels (Figure 2(E)), and inhibition of miR‐409‐3p enhanced the expression of CTRP3 in PC12 cells (Figure 2(F)).
Enlarge this image.2 FIGURE. CTRP3 was a target gene of miR‐409‐3p. (A) According to TargetScan, a conserved binding site was found in the 3'UTR of CTRP3. (B) the dual luciferase reporter assay indicated that miR‐409‐3p significantly suppressed the relative luciferase activity of pmirGLO‐CTRP3‐3'UTR. (C) RT‐PCR analysis demonstrated that transfection of the miR‐409‐3p mimic significantly enhanced the miR‐409‐3p level. (D) Western blot assay indicated that miR‐409‐3p significantly suppressed the expression of CTRP3 in PC12 cells. (E) Transfection of miR‐409‐3p inhibitor significantly suppressed miR‐409‐3p levels. (F) Inhibition of miR‐409‐3p enhanced the expression of CTRP3 in PC12 cells. **p < 0.01, ***p < 0.001 vs. controls
Previous studies have indicated that knockout of CTRP3 induced cell apoptosis by activating JNK/p38 signaling.19,20 Hence, we explored the effect of miR‐409‐3p on PC12 cell apoptosis. Our data showed that transfection with miR‐409‐3p mimic induced activation of JNK/p38 signaling and increased the expression of apoptosis‐related proteins, including Bax and c‐caspase 3 (Figure 3(A)). TUNEL staining showed that overexpression of miR‐409‐3p obviously enhanced cell apoptosis in PC12 cells (Figure 3(B)).
Enlarge this image.3 FIGURE. Reduced CTRP3 led to PC12 cell apoptosis by activating JNK/P38 signaling in PC12 cells. (A) Transfection with miR‐409‐3p mimic induced activation of JNK/p38 signaling and increased the expression of apoptosis‐related proteins, including Bax and c‐caspase 3, in PC12 cells. (B) TUNEL staining showed that overexpression of miR‐409‐3p obviously enhanced cell apoptosis in PC12 cells. *p < 0.05, **p < 0.01, ***p < 0.001 vs. controls
To further elucidate whether miR‐409‐3p induced cell apoptosis via CTRP3, a specific siRNA targeting CTRP3 was selected. Western blot analysis showed that silencing CTRP3 significantly increased the expression of p‐JNK, p‐p38, Bax and c‐caspase 3 even in PC12 cells transfected with miR‐409‐3p inhibitor (Figure 4(A)). Moreover, flow cytometry analysis also indicated that miR‐409‐3p inhibition resulted in much lower cell apoptosis (Figure 4(B)). However, such effects could be significantly abolished by silencing CTRP3 (Figure 4(B)). These data indicated that miR‐409‐3p‐induced cell apoptosis was mediated via CTRP3 in PC12 cells.
Enlarge this image.4 FIGURE. miR‐409‐3p‐induced cell apoptosis was mediated via CTRP3 in PC12 cells. (A) Western blot analysis showed that silencing CTRP3 significantly increased the expression of p‐JNK, p‐p38, Bax and c‐caspase 3 even in PC12 cells transfected with miR‐409‐3p inhibitor. (B) Flow cytometry analysis also indicated that miR‐409‐3p inhibition resulted in much lower cell apoptosis, but such effects could be significantly abolished by silencing CTRP3. *p < 0.05, **p < 0.01, ***p < 0.001 vs. as indicated
Finally, we determined the level of serum CTRP3 in patients with ACI. Compared with healthy controls, the level of serum CTRP3 was significantly decreased in patients with ACI (Figure 5(A)). Moreover, serum CTRP3 was also found to be gradually reduced in patients in the large infarction group compared with those in the middle infarction group and small infarction group (Figure 5(B)). Additionally, much lower levels of serum CTRP3 were demonstrated in patients with ACI in the middle group and severe group than in the mild group (Figure 5(C)), indicating that lower serum CTRP3 correlated with impaired cerebral function. Pearson correlation analysis showed that serum CTRP3 negatively correlated with plasma miR‐409‐3p in patients with ACI (r = −0.750, p < 0.001) (Figure 5(D)).
Enlarge this image.5 FIGURE. Serum CTRP3 negatively correlated with plasma miR‐409‐3p in patients with ACI. (A) Compared with healthy controls, the level of serum CTRP3 was significantly decreased in patients with ACI. (B) Serum CTRP3 was also found to be gradually reduced in patients in the large infarction group compared with those in the middle infarction group and small infarction group. (C) Much lower levels of serum CTRP3 were found in patients with ACI in the middle group and the severe group than in the mild group. (D) Pearson correlation analysis showed that serum CTRP3 negatively correlated with plasma miR‐409‐3p in patients with ACI. **p < 0.01, ***p < 0.001 vs. as indicated
ACI is a major cause of death and an important contributor to brain‐related mortality worldwide.21 Increasing evidence suggests that circulating microRNAs may be potential biomarkers for the diagnosis of ACI.21–23 For instance, miR‐124 levels are shown to be reduced in ACI patients and the dynamic changes of miR‐124 may be useful for the monitor of ischemic stroke.24
In the present study, we found that the expression of plasma miR‐409‐3p in the ACI group was higher than that in healthy controls. Furthermore, the subjects were divided into four groups according to infarct volume and NIHSS score. The results showed that the relative expression of plasma miR‐409‐3p gradually increased with impaired cerebral function. Pearson correlation analysis showed that the expression of plasma miR‐409‐3p was positively correlated with the NIHSS score, which further suggested that plasma miR‐409‐3p could be used to evaluate the severity of ACI. Some factors, including low‐density lipoprotein cholesterol (LDL‐C), obesity, diabetes, hypertension, and age, are suggested to be traditional risk factors for ACI.25 However, we found plasma miR‐409‐3p demonstrated no significant correlation with these risk factors, indicating that miR‐409‐3p may affect the progression of ACI through other mechanisms. ROC analysis indicated that plasma miR‐409‐3p may be a potential biomarker to screen ACI patients from healthy controls. Based on the above data, we proposed that enhanced plasma miR‐409‐3p may be a risk factor in the progression of ACI. To our knowledge, this is the first study which indicates the important role of plasma miR‐409‐3p in patients with ACI.
Then, we explored the possible target genes of miR‐409‐3p. Interestingly, CTRP3, a novel adipose tissue‐derived secreted factor, was found to be a target gene of miR‐409‐3p. CTRP3 has been shown to exert beneficial biological effects on metabolism, inflammation, and survival signaling in multiple tissues.26 Previous studies indicate that circulating CTRP3 levels are decreased in patients with cardiovascular events such as obesity and metabolic syndrome.27,28 For instance, CTRP3 is reported to enhance the contraction of cardiomyocytes by elevating myofilament Ca2+ sensitivity, which then modulates the contractility of cardiomyocytes in mice.29 In ischemic mice, the antiapoptotic, proangiogenic, and cardioprotective roles of CTRP3 have been determined.30 In addition, silencing of CTRP3 induced cell apoptosis by activating JNK/p38 signaling in mice with depression.19,20 Here, the effect of miR‐409‐3p on the apoptosis of PC12 cells was evaluated. Our data showed that transient overexpression of miR‐409‐3p significantly increased cell apoptosis. In consistent, knockdown of CTRP3 activated the JNK/p38 signaling and enhanced PC12 cell apoptosis, but such effect could be reversed by inhibition of miR‐409‐3p. These results indicated that miR‐409‐3p may promotes the the progression of ACI through enhancing the apoptosis of neuron cells.
Furthermore, we evaluated the expression of serum CTRP3 in patients with ACI, and our data showed that serum CTRP3 was decreased in patients with ACI compared with healthy controls. In addition, decreased serum CTRP3 was correlated with decreased cerebral function. These findings were in line with previous studies.31,32 In rats, CTRP3 has been shown to function as a neuroprotective adipokine by regulating the MPK/HIF‐1α/VEGF‐dependent pathway.31 During intracerebral hemorrhage, CTRP3 may modulate oxidative stress injury via PKA signaling.32 Hence, reduced serum CTRP3 is highly associated with cerebral injury and may affect the progression of brain function in the blood circulation of patients. We propose that miR‐409‐3p may play a key role in cerebral tissue injury and pathological processes via different means. However, large experiments are needed to verify the hypothesis.
There are limitations in the present study. First, large patient samples are required to validate the present findings. In addition, whether the combined use of plasma miR‐409‐3p and other indicators would increase the substantial diagnostic value needs to be evaluated in detail. Third, it is interesting to explore the serum levels of miR‐409‐3p and CTRP3 in >24 h after stroke. This will help us to understand the dynamic changes of miR‐409‐3p and ctrp3 in serum, thus providing a new target for the treatment of ACI.
In summary, for the first time, we showed novel data that elevated plasma miR‐409‐3p is correlated with disease severity and may be effective for the early diagnosis of ACI.
All authors declare no conflict of interest.
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1 Department of orthopedics, Hongqi Hospital Affiliated to Mudanjiang Medical University, Mudanjiang, Heilongjiang, China
2 Department of Radiology, Hongqi Hospital Affiliated to Mudanjiang Medical University, Mudanjiang, Heilongjiang Province, China