BV treatment relieves cerebral infarction and decreases the levels of serum IL-1β, IL-6 and TNF-α
TTC staining was used to detect the cerebral infarction volume at 24h after reperfusion to verify whether BV administration could improve functional deficits in rats with CIR. We found that the infarct volume percentage (%) in IR group was significantly larger than that in Sham group (P<0.05, Fig. 1A and 1B). The infarct volume percentage (%) in BV group was obviously reduced than that in IR group (P<0.05, Fig. 1B), suggesting that BV could relieve cerebral infarct caused by cerebral ischemia reperfusion injury. According to the results of ELISA, the rats in IR group had a higher levels of IL-1β, IL-6 and TNF-α in the serum than those in the Sham group, while the levels of IL-1β, IL-6 and TNF-α in the serum in BV group were evidently decreased compared with those in IR group (P<0.05, Fig. 1C).
BV treatment induced abnormal microRNAs expression
We used microRNA microarray analysis to detect the expression profiles of miRNAs in cortex tissues isolated from rats in Sham, IR and BV groups in our previous study. According to the results of miRNA microarray, we selected the top 10 differentially expressed miRNAs to perform for heatmap (Fig. 2A). miRWalk, miRDB and TargetScan databases were used to predict the targeted genes of miR-27a-3p. There are 838, 667, 990 targeted genes of miR-27a-3p from miRWalk, miRDB and TargetScan databases (Fig. 2B), among them, 42 genes are common. Then, Cytoscape was used to draw interaction network between miR-27a-3p and its common targeted genes (Fig. 2C).
GO and KEGG pathway analysis of targeted genes of miR-27a-3p
To explicit the biological function of miR-27a-3p, we performed for GO analysis of 42 targeted genes of miR-27a-3p. We found that the top 10 biological processes (BP) of 42 targeted genes of miR-27a-3p were cellular macromolecule metabolic process, cellular protein modification process, cellular response to stress, macromolecule modification, positive regulation of biological process, positive regulation of cellular process, protein modification process, regulation of nucleobase containing compound metabolic, regulation of striated muscle tissue development and response to stress. The top 10 cellular component (CC) were intracellular membrane bounded organelle, intracellular organelle, intracellular organelle lumen, membrane bounded organelle, membrane enclosed lumen, nuclear lumen, nuclear part, nucleus, organelle and organelle lumen. The top 10 molecular function (MF) were kinase activity, phosphotransferase activity, protein binding, protein kinase activity, receptor activator activity, receptor agonist activity, receptor regulator activity, transcription factor activity, transcription factor activity and protein binding and transferase activity (Fig. 3A).
To reveal the pathways of miR-27a-3p, we performed for KEGG pathway analysis of 42 targeted genes of miR-27a-3p. Results showed that the top 5 pathways of 42 targeted genes of miR-27a-3p were p53 signaling pathway, Wnt signaling pathway, mTOR signaling pathway, cGMP-PKG signaling pathway and regulation of autophagy (Fig. 3B). Among them, the targeted gene LITAF of miR-27a-3p mainly involved in p53 signaling pathway and regulation of autophagy.
LITAF as a target of miR-27a-3p
The target sites of miR-27a-3p in the 3’UTR of LITAF were shown in Fig. 4A. In order to experimentally confirm information, portions of the 3’UTR of these potential targets were cloned into pGL3 control vector, downstream of luciferase-coding sequences. The recombinational plasmids of 3’UTR of LITAF-WT or 3’UTR of LITAF-MUT were co-transfected with miR-27a-3p or miR-27a-3p (mimic NC) and Renilla into cells. The relative luciferase activity was decreased in cells co-transfected with miR-27a-3p and 3’UTR of LITAF compared with cells co-transfected with mimic NC and 3’UTR of LITAF, there is no significant difference in relative luciferase activity between cells co-transfected with mimic NC and 3’UTR of LITAF-MUT compared with cells co-transfected with miR-27a-3p and 3’UTR of LITAF-MUT (Fig. 4B). These results suggested that miR-27a-3p can negatively regulate LITAF.
Temporal expression of miR-27-3p and LITAF after CIR
The CIR-induced changes in the miR-27-3p and LITAF expression levels were examined for 24 h post-surgery. MiR-27-3p expression was obviously downregulated with time and reached its lowest levels at both 24 and 48 h after CIR compared with the levels in the Sham group (Fig. 5A, P < 0.05). Likewise, the LITAF expression levels were significantly increased beginning from 6 h after IR, and this high level reached its highest levels at 24 and 48h after CIR maintained throughout the observation period (Fig. 5B, P < 0.05), suggesting a potential negative correlation between miR-27-3p and LITAF expression (Fig.5C).
Expression levels of miR-27a-3p and LITAF in cortex of rat transfected with lentivirus
Seven day after transfected lentiviru, qRT-PCR was used to detect relative expression levels of miR-27a-3p and Litaf in rats. We found that relative expression level of miR-27a-3p was increased in miR-27a-3p mimic group (P<0.01, Fig. 6A), while decreased in miR-27a-3p inhibitor group (P<0.05, Fig. 6A). Moreover, to investigate the effect of overexpression of Litaf on miR-27a-3p, we injected Litaf mimic at the same. We found that relative expression of Litaf was increased in Litaf mimic group (Fig. 6B).
MicroRNA-27a-3p relives neurologic deficit after cerebral ischemia reperfusion
To determine the effect of miR-27-3p on the neurological function in rats with CIR, NSS was used to assess the functional recovery. The NSS test was performed at 6h, 12h, 24h and 48h post‑reperfusion in Sham, IR, IR+NC, IR+miR-27a-3p mimic, IR+miR-27a-3p inhibitor and IR+miR-27a-3p mimic + Litaf mimic groups. We found that NSS score showed similar tendency in six groups at four sampling time (Fig. 7A). We focus the NSS scores in six groups at 24h post-reperfusion. Compared with the Sham group, functional deficits were impaired by ischemic insult in the IR group at 6h, 12h, 24h and 48h post‑reperfusion (P<0.01; Fig. 7B). A slight recovery of neurological functions was observed in the IR+miR-27a-3p mimic group, while a slight exacerbation of neurological functions was found in the IR+miR-27a-3p inhibitor and IR+miR-27a-3p mimic + Litaf mimic groups at 6h, 12h, 24h and 48h post‑reperfusion (Fig. 7B). Therefore, it can be concluded that miR-27a-3p mimic effectively decreased cerebral infarction volume and may improve functional recovery.
MiR-27a-3p mimic relieves cerebral infarct induced by CIR
TTC staining was used to detect the cerebral infarction volume at 24h after reperfusion to verify whether miR-27a-3p mimic administration could improve functional deficits in rats with CIR. We found that the infarct volume percentage (%) in IR group was significantly larger than that in Sham group (P<0.05, Fig. 8A and 8B). The infarct volume percentage (%) in IR+miR-27a-3p mimic group was obviously reduced than that in IR group (P<0.05, Fig. 8B), while in IR+miR-27a-3p inhibitor was increased than that in IR group, suggesting that miR-27a-3p overexpression could relieve cerebral infarct caused by cerebral ischemia reperfusion injury.
MiR-27a-3p mimic inhibits the fluorescence signal for LITAF in neuron and microglial cell
As shown in Fig. 9A and 9B, the majority of the fluorescence signal for LITAF in the IR group was localized in the cells positive for NeuN and Iba1 (cells with yellow signals) at both 24 h after IR. Representative photomicrographs and quantification showed that miR-27a-3p mimic injection significantly decreased LITAF immunoreactivity and the number of LITAF-positive double-labeled cells in the neurons and microglia (Fig. 9A, 9B and 9C, P<0.05), while increased LITAF immunoreactivity and the number of LITAF-positive double-labeled cells was observed with miR-27a-3p inhibitor injection, no such change was observed with miR-27a-3p control injection (IR+NC group). These effects in response to miR-27a-3p mimic were also increased by the addition of LITAF mimic (Fig. 9A, B, C, P < 0.05). No significant differences were found between the IR and IR+NC groups (P > 0.05).
MiR-27a-3p mimic deceases neuronal damage induced by CIR
After 24h of reperfusion, Nissl staining was employed to exhibit morphological alterations in cortex. Normal neurons were polygonal and the nissl bodies were blue patches in the cytoplasm in Sham group (Fig. 10A), while nissl bodies were dissolved in IR group (Fig. 10A). However, in IR+miR-27a-3p mimic group, these changes were rare (Fig. 10A). Neurons count in cortex examined the quantity of positive stained neurons (Fig. 10B). Compared with Sham group, surviving neurons count of IR declined significantly. Surviving neurons were increased obviously after injection of miR-27a-3p mimic, while decreased obviously after injection of miR-27a-3p inhibitor (Fig.10B).
MiR-27a-3p modulate Litaf to release IL-6
Double immunofluorescence analysis was used to verify whether miR-27a-3p could regulate Litaf to release IL-6. We found that Litaf/IL-6 were both co-expressed at 24h after reperfusion in cortex of rats (Fig. 11A). Litaf/IL-6 double positive cells were increased in IR and IR+NC groups compared with those in Sham group (P<0.05) (Fig. 12B and C). After addition of miR-27a-3p mimic, Litaf/IL-6 double positive cells were decreased than those in IR and IR+NC group (P<0.05, Fig. 12B and C). Litaf/IL-6 double positive cells in IR+miR-27a-3p mimic+Litaf mimic groups were increased compared with those in IR+miR-27a-3p group (Fig. 11B and C). Western blot was used to detect the effect of miR-27a-3p on the protein expression of IL-6. We found that the relative protein expression levels of IL-6 were increased in IR and IR+NC groups compared with these in Sham group (P<0.05, Fig. 11D-E). While, after injection of miR-27a-3p mimic, relative protein expression levels of IL-6 were decreased in IR+miR-27a-3p compared with these IR and IR+NC group (P<0.05, Fig. 11D-E). ELISA results showed that serum IL-6 level was increased in IR and IR+NC group than that in Sham group (P<0.05, Fig. 12F). After addition of miR-27a-3p inhibitor, serum IL-6 level was increased than that in IR and IR+NC group (P<0.05, Fig. 11F), the same result was seen in IR+miR-27a-3p mimic+Litaf mimic group (P<0.05, Fig. 11F).
MiR-27a-3p regulates Litaf through TLR4/NF-κB
Double immunofluorescence analysis was used to verify whether miR-27a-3p could regulate Litaf though TLR4/NF-κB signal pathway. We found that Litaf/TLR4, Litaf/ NF-κB were both co-expressed (Fig. 12A-B). Western blot was used to detect the effect of miR-27a-3p on the protein expression of Litaf, TLR4, NF-κB, p-p65 and p65. We found that the relative protein expression levels of Litaf, TLR4 and p-p65 were increased in IR and IR+NC groups compared with these in Sham group (P<0.05, Fig. 12C-D). While, after injection of miR-27a-3p mimic, relative protein expression levels of Litaf, TLR4 and p-p65 were decreased in IR+miR-27a-3p compared with these IR and IR+NC group (P<0.05, Fig. 12C-D).