miR-212-5p levels are downregulated in individuals with ischemic stroke
RNA-seq was performed to detect the expression profiles of miRNAs in the blood of AIS patients (n=3) and healthy controls (HC) (n=3) and in the blood and brain of rats in the MCAO group (n=3) and the sham group (n=3). A total of 508 miRNAs were found to be significantly differentially expressed between AIS patients and healthy controls. Compared with sham group, miRNA expression profiles also exhibited significant changes in blood and the peri-infarct area in rats 72 h after MCAO, 34 miRNAs were significantly differentially expressed in rat blood between the MCAO group and the sham group and 87 miRNAs were significantly differentially expressed in rat brains between the MCAO group and the sham group. The threshold set for significantly differentially expressed miRNAs was an absolute value of fold
change of more than 2.0, | log2 fold change | more than 1 and a P value of less than 0.05. A heatmap (Fig. 1A, B, C) and volcano plot (Fig. 1D, E, F) were used to visualize the differences in the miRNA expression levels in different groups. RNA-seq revealed that the expression of miR-212-5p was downregulated in the blood of patients with AIS, and in the brain and blood of MCAO rats.
RT–qPCR results showed that the expression level of miR-212-5p was significantly decreased in AIS patients compared to that in healthy controls (p<0.05, Fig. 1G). The results also showed that the expression level of miR-212-5p was significantly reduced in both the blood (p<0.05, Fig. 1H) and brain (p<0.05, Fig. 1I) of rats after MCAO compared with the findings in the sham group, which is consistent with the change in miR-212-5p level identified by miRNA RNA-seq.
Receiver operator characteristic (ROC) curve analysis was performed, and the area under the ROC curve (AUC) was calculated to investigate the diagnostic value of miR-212-5p for AIS. As shown in Fig. 1, the AUC was 0.96 (95% CI =0.92 to 1.00) for AIS in human blood (Fig. 1J), 0.95 (95% CI =0.90 to 1.00) for MCAO rats in blood (Fig. 1K) and 0.93 (95% CI =0.86 to 1.00) for MCAO rats in brain (Fig. 1L); the result of ROC curve analysis indicated that blood miR-212-5p has certain diagnostic value for AIS and indicated that changes in miR-212-5p expression in rat blood and brain were valuable to distinguish the MCAO group from the sham group.
Our results showed that the expression of miR-212-5p was decreased in the blood of AIS patients and in both the blood and brain of MCAO rats, and it also has diagnostic significance for AIS, moreover, previous studies have suggested that it is closely related to the development of AIS. Thus, we selected it for further study.
Delineation of GO and KEGG pathway analysis
We performed GO and KEGG pathway analysis of the putative target genes to infer the potential functions of significantly differentially expressed miRNAs. GO analysis was conducted based on the predicted target genes of the differentially expressed miRNAs to identify the enriched molecular functions (MFs), cellular components (CCs) and biological processes (BPs). The representative GO terms significantly enriched by predicted target genes included RNA binding, response to oxidative stress, calcium channel activity, regulation of neurogenesis and neuron apoptotic process (Fig. 2A, B, C), while KEGG pathway analysis showed that some significantly enriched signaling pathways may be involved in the pathological mechanisms of ischemic brain damage, including apoptosis, calcium signaling pathway, neurotrophin signaling pathway, TNF signaling pathway, platelet activation and VEGF signaling pathway (Fig. 2D, E, F). The results of GO enrichment and KEGG pathway analysis showed that the biological functions of the predicted target genes in patients with AIS and in rats with ischemic stroke were similar. This finding suggests the feasibility of the MCAO model for studying the pathological mechanism of AIS.
Lncfos directly binds to miR-212-5p, and CASP7 is the direct target of miR-212-5p
The specific binding sites between CASP7 and miR‐212‐5p were predicted by bioinformatics software (Fig. 3A). Moreover, online prediction analysis software predicted a relationship between miR‐212‐5p and lncfos (Fig. 3B, E). Subsequently, the luciferase reporter assay system was used to verify whether CASP7 is the target gene of miR-212-5p. Our results showed that compared with the NC group, the CASP7-WT+miR-212-5p group exhibited significantly decreased luciferase activity intensity (Fig. 3C), but the intensity remained almost unchanged in the CASP7-MUT+miR-212-5p group (Fig. 3C), suggesting that miR-212-5p directly targets CASP7 by binding to the CASP7 3’-UTR in the predicted binding site. Additionally, we revealed that when compared with the mimic NC group, the relative intensity of luciferase activity in the lncfos-WT+miR‐212‐5p mimic group decreased noticeably (P<0.05, Fig. 3D), while in the lncfos‐MUT+miR‐212‐5p mimic group, it remained almost unchanged (P>0.05, Fig. 3D), indicating that lncfos is a direct target gene of miR‐212‐5p.
Successful establishment of the MCAO model and accurate localization of the right lateral ventricle
In this research, MCAO was conducted to establish a rat model of ischemic stroke. Symptoms of neurological deficit, neurological function deficit scores and HE staining were used to ensure the successful establishment of the rat MCAO model and the accurate localization of the right lateral ventricle. When compared with the sham group, contralateral forepaw flexion was observed in the MCAO group (Fig. 3F). The neurological function deficit scores shown in Fig. 3H indicated that the neurological function deficit score of the MCAO group was noticeably increased compared with that of the sham group. HE staining showed accurate localization of the right lateral ventricle (Fig. 3G). Our results also showed that overexpression or inhibition of miR-212-5p was achieved by intracerebroventricular administration of miR-212-5p agomir or miR-212-5p antagomir (Fig. 3I). The aforementioned experimental results demonstrated the successful establishment of rat models of brain ischemic stroke and accurate localization of the right lateral ventricle.
Silencing lncfos or overexpressing miR-212-5p protects against MCAO-induced brain damage in rats
Overexpression of miR-212-5p or lncfos silencing in rats diminished cerebral infarction (Fig. 4A, B), alleviated pathological damage (Fig. 4C), increased the number of viable neurons (Fig. 4D, E), improved the survival rate of rats (Fig. 4F) and ameliorated neurological deficits (Fig. 4G) following MCAO compared with the effects observed in the MCAO group.
The results of TTC staining showed that upregulation of miR-212-5p by injection with miR-212-5p agomir or downregulation of lncfos by injection with sh-fos reduced the infarct size of rat brain tissues in rat MCAO models. In comparison with the MCAO group, the sh-fos+miR-212-5p antagomir group or the agomir NC group, the miR-212-5p agomir group exhibited a significant decrease in cerebral infarct size (p< 0.05; Fig. 4A, B), while there was no significant difference between the MCAO group and the agomir NC group (p>0.05; Fig. 4A, B).
The HE staining results showed that the majority of the neuronal cells in the MCAO group, sh-fos+miR-212-5p antagomir group and agomir NC group displayed hydroncus, karyopyknosis and necrosis. In the sham group, the sh-fos+miR-212-5p antagomir NC group and miR‐212 agomir group showed a decreased number of neuronal cells with karyopyknosis and necrosis (Fig. 4C).
Nissl staining was also conducted to assess neuronal damage in MCAO rats in each group. In the sham group, the neurons in the peri-infarct region remained intact, while in the MCAO group, sh-fos+miR-212-5p antagomir group and agomir NC group, diffuse neuronal degeneration and necrosis were observed (Fig. 4D). The numbers of viable neurons in the sham group, MCAO group, sh-fos+miR-212-5p antagomir group, sh-fos+antagomir NC group, miR-212-5p agomir and agomir NC
group were compared (Fig. 4D). A significant decrease in the number of viable neurons was seen in the MCAO group, sh-fos+miR-212-5p antagomir group and agomir NC in comparison to the sham group (p<0.05, Fig. 4E). In the sh-fos+antagomir NC group and miR-212-5p agomir group, the number of viable cells increased significantly compared with that in the MCAO group (Fig. 4E). In addition, the number of viable cells in the miR-212-5p agomir group was markedly higher than that in the sh-fos+miR-212-5p antagomir group (p<0.05, Fig. 4E). Each count was repeated three times.
Neurological deficiency scores were evaluated before MCAO and at 1, 3, 5, 7, and 14 days after MCAO and treatment. The mNSS scores showed that at 3, 5, 7, and 14 days after MCAO, the mNSS score of the miR-212-5p agomir group was significantly decreased compared with that of the sh-fos+miR-212-5p antagomir group (p < 0.05, Fig. 4G), and the mNSS score in the sh-fos+miR-212-5p antagomir NC group decreased after upregulating miR-212-5p compared to that in the MCAO or agomir NC group. When compared with the sham group, all the other groups had significantly higher mNSS scores at each time point (Fig. 4G). The results indicated that the neurological function of MCAO rats recovered to some extent after sh-fos+miR-212-5p antagomir NC injection along with significant improvement by upregulating miR-212-5p expression via miR-212-5p agomir injection.
Silencing of lncfos or overexpression of miR-212-5p attenuates neuronal cell apoptosis by inhibiting CASP7 in MCAO rats
TUNEL staining was performed to evaluate apoptosis in peri-infarct area neurons. The normal neuronal karyon was stained blue. Apoptotic cell nuclei were stained fluorescent green, and karyopyknosis was irregular in shape. Representative TUNEL- positive cells in the peri-infarct area in each group are shown in Fig. 5A. Compared with the sham group, the numbers of TUNEL-positive cells in the peri-infarct area were significantly increased in the MCAO, sh-fos+miR-212-5p antagomir and agomir NC groups (p<0.05, Fig. 5A, C). Compared with the MCAO group, the miR-212-5p agomir group had a lower number of TUNEL-positive cells in the peri-infarct area; however, the sh-fos+miR-212-5p antagomir group had an elevated number of TUNEL-positive cells in the peri-infarct area (p<0.05, Fig. 5A, C).
The immunohistochemistry results showed that the integrated optical density (IOD) value of CASP7 was much lower in the sham group than in the MCAO group. The IOD value of CASP7 was decreased after overexpression of miR-212-5p or downregulation of lncfos. In comparison with the sh-fos+miR-212-5p antagomir group, the miR-212-5p agomir group had a lower IOD value of CASP7 (Fig. 5B, D), while there was no significant difference between the MCAO group and agomir NC group (Fig. 5B, D).
Further study showed that lncfos expression was increased in the brain of MCAO rats, and could be effectively silenced by intracerebroventricular injection of sh-fos. The PCR results showed that the relative expression level of lncfos was significantly decreased in the sh-fos group compared with the MCAO group and the sh-fos NC group (p<0.05, Fig. 5E).
The aforementioned study results indicated that the pathological conditions of the rats subjected to MCAO recovered to some extent after sh-fos injection and were significantly improved by overexpression of miR-212-5p and then downregulation of CASP7 via the lncfos/miR-212-5p/CASP7 axis and probably through the apoptosis signaling pathway.
Silencing lncfos and increasing miR‐212‐5p expression decreased CASP7 expression in the brains of MCAO rats
First, we evaluated the effect of lncfos on the expression of miR-212-5p. The expression of miR-212-5p in the peri-infarct area was measured after intracerebroventricular injection of sh-fos, sh-NC, sh-fos+miR-212-5p antagomir and sh-fos+antagomir NC. The results showed that the relative expression level of miR-212-5p was significantly increased in the sh-fos group compared with the sh-NC group, while miR-212-5p expression was noticeably decreased in the sh-fos+miR-212-5p antagomir group compared with the sh-fos group (p<0.05, Fig. 6A); however, there was no significant difference between the sh-fos and sh-fos antagomir NC groups (p>0.05, Fig. 6A). The above results indicated that lncfos directly binds to miR-212-5p and affects the expression of miR-212-5p in rat MCAO models.
PCR analysis showed that the relative mRNA CASP7 expression significantly increased in rat brains after MCAO compared with the expression in the sham group (P<0.05, Fig. 6B). Compared to the MCAO or sh-fos+miR-212-5p antagomir group, the miR-212-5p agomir group had significantly decreased CASP7 mRNA expression, while the sh-fos+antagomir NC group had significantly lower CASP7 mRNA expression than the sh-fos+miR-212-5p antagomir group (P < 0.05, Fig. 6B).
Western blot results showed that the expression level of CASP7 was significantly elevated in the MCAO group compared to the sham group, and after intracerebroventricular injection of miR-212-5p agomir, the expression level of CASP7 was significantly decreased when compared to that in the MCAO group or sh-fos+miR-212-5p antagomir group (Fig. 6 C, D).
Immunofluorescence staining was also performed to examine the expression of CASP7 in the peri-infarct area of MCAO rats (Fig. 6E, F). The results demonstrated that the expression of CASP7 in the peri-infarct area of the MCAO group, sh-fos+miR-212-5p antagomir group and agomir NC group was significantly elevated compared with that in the sham group (Fig. 6E, F), while compared with that in the miR-212-5p agomir group, the expression level of CASP7 was significantly increased in the MCAO group or sh-fos+miR-212-5p antagomir group (Fig. 6 E, F).
In this study, we provided evidence to confirm that miR-212-5p ameliorated MCAO-induced neuronal apoptosis through regulation of the lncfos/miR-212-5p/CASP7 axis (Fig. 6G).