MiR-27a-3p suppresses cerebral ischemia-reperfusion injury by targeting FOXO1

OGD-induced inhibition of HT22 cell growth is significantly reversed by miR-27a-3p mimics

To mimic CI/R injury in vitro, the oxygen-glucose deprivation (OGD) cell model was established. As indicated in Figure 1A, OGD significantly inhibited miR-27a-3p expression in HT22 cells, and this inhibitory effect was reversed by miR-27a-3p mimics. In addition, the viability of HT22 cells was significantly reduced by OGD, while this effect, too, was significantly reversed by miR-27a-3p mimics (Figure 1B). Meanwhile, miR-27a-3p mimics alone limitedly affected the viability of HT22 cells (Figure 1B). Consistent, with those findings, the antiproliferative effect of OGD on HT22 cells was significantly reversed by miR-27a-3p mimics (Figure 1C, 1D). All of these data suggested that OGD-induced HT22 cell growth inhibition is reversed by miR-27a-3p mimics.

Figure 1. OGD-induced HT22 cell growth inhibition is significantly reversed by miR-27a-3p mimics. HT22 cells were transfected with mimics-control or miR-27a-3p mimics and then treated with OGD. (A) Expression of miR-27a-3p in HT22 cells detected with q-PCR. (B) Viability of HT22 cells assessed with CCK-8 assays. (C) Ki67 staining (red) showing proliferation of HT22 cells. Nuclei were counterstained with DAPI (blue). (D) Rate of Ki67 positivity. ^**P<0.01 vs. control. ^##P<0.01 vs. OGD.

OGD-induced cell apoptosis is reversed by miR-27a-3p mimics

Using flow cytometry to detect cell apoptosis, we observed that OGD clearly induced apoptosis in HT22 cells (Figure 2A, 2B). Consistent with the increased apoptosis, levels of active caspase 3 and active caspase 9, two pro-apoptosis enzymes, were significantly increased in HT22 cells in the presence of OGD (Figure 2C–2E). Conversely, levels of the anti-apoptotic protein (Bcl-2) was significantly decreased in HT22 cells in the presence of OGD (Figure 2C, 2F). All of these effects were reversed in the presence of miR-27a-3p mimics (Figure 2A–2F).

Figure 2. OGD-induced cell growth inhibition is reversed by miR-27a-3p mimics. (A) FACS analysis of cell apoptosis. (B) The rate of apoptosis among HT22 cells. (C) Western blotting showing levels of active caspase 3, active caspase 9 and Bcl-2 in HT22 cells. (D–F) Relative protein expression levels of active caspase 3 (D), active caspase 9 (E) and Bcl-2 (F) normalizing to GAPDH. ^**P<0.01 vs. control. ^##P<0.01 vs. OGD.

MiR-27a-3p reverses OGD-induced injury in HT22 cells

Using flow cytometry to detect levels of reactive oxygen species (ROS) in HT22 cells, we found that OGD upregulated ROS levels in HT22 cells (Figure 3A, 3B). OGD also significantly reduced the levels of the antioxidants glutathione and superoxide dismutase and increased the levels of malondialdehyde, a marker of oxidative stress, in the supernatants of HT22 cells (Figure 3C–3E). Again, all of these effects were at least partially reversed in the presence of miR-27a-3p mimics (Figure 3A–3E). Thus, miR-27a-3p mimics clearly suppressed OGD-mediated oxidative injury in HT22 cells.

Figure 3. MiR-27a-3p protects against OGD-induced injury in HT22 cells. (A) FACS analysis of ROS level in HT22 cells. (B) Relative levels of ROS. (C–E) Glutathione (GSH) (C), malondialdehyde (MDA) (D) and superoxide dismutase (SOD) levels (E) in detected in supernatants of HT22 cells with ELISAs. ^**P<0.01 vs. control. ^#P<0.05, ^##P<0.01 vs. OGD.

MiR-27a-3p directly targets FOXO1

The targetscan (http://www.targetscan.org/vert_71/), miRDB (http://www.mirdb.org/) and miRWalK (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/micrornapredictedtarget.html) on-line tools were used to explore the gene targeted by miR-27a-3p. As shown in Figure 4AFOXO1 was the likely target of miR-27a-3p. This was confirmed by dual luciferase assays, which showed the suppression of FOXO1 transcription by miR-27a-3p (Figure 4B). Additionally, RT-qPCR demonstrated that miR-27a-3p mimics significantly reversed OGD-induced increases in FOXO1 gene expression in HT22 cells (Figure 4C).

Figure 4. MiR-27a-3p directly targets FOXO1 in HT22 cells. (A) Gene structure of FOXO1 at position 426-432 indicates the predicted target binding site of miR-27a-3p in its 3'UTR. (B) Luciferase activity measured after co-transfecting with wild-type or mutant FOXO1 3′-UTR plasmid and miR-27a-3p mimics into HT22 cells. The results were normalized to Renilla luciferase activity. (C) RT-qPCR analysis of FOXO1 expression in HT22 cells. ^**P<0.01 vs. control. ^##P<0.01 vs. OGD.

MiR-27a-3p reverses OGD-induced G1 arrest in HT22 cells via mediation of FOXO1/p27 Kip1 axis

We used western blotting to explore the mechanism by which OGD mediates the progression of CI/R in vitro. As shown in Figure 5A–5C, OGD upregulated protein expression of FOXO1 and p27 Kip1, the cell cycle mediator [14], in HT22 cells, while this phenomenon was suppressed in the presence of miR-27a-3p mimics. Moreover, flow cytometric analysis confirmed that OGD induced G1 arrest in HT22 cells, while overexpression of miR-27a-3p partially reversed the effect of OGD on cell cycle distribution (Figure 5D, 5E). These results suggest that OGD induces G1 arrest in HT22 cells via FOXO1/p27 Kip1 signaling, and that effect is reversed by miR-27a-3p.

Figure 5. MiR-27a-3p reverses OGD-induced G1 arrest in HT22 cells via mediation of FOXO1/p27 Kip1 axis. (A) Western blotting showing the protein expression of FOXO1 and p27 Kip1 in HT22 cells. (B, C) Relative expression levels of FOXO1 (B) and p27 Kip1 (C) normalized to GAPDH. (D) FACS analysis of HT22 cell cycling after PI staining. (E) Cell cycle profile showing distribution of cells in G0/G1, S or G2 phase. ^**P<0.01 vs. ^#P<0.05, ^##P<0.01 vs. OGD.

MiR-27a-3p agomir significantly attenuates the symptom of CI/R in vivo

Finally, a rat model of CI/R was established to explore the effect of miR-27a-3p on CI/R progression in vivo. As indicated in Figure 6A, 6B, miR-27a-3p agomir significantly reduced cerebral infraction area in CI/R rats. In addition, miR-27a-3p agomir reversed CI/R-induced increases of cerebral water content and the TUNEL positivity rate (Figure 6C, 6D). Consistent with our in vitro findings, CI/R induced increases in FOXO1, p27 Kip1 and active caspase 3 in brain tissues of the model rats, but those effects were significantly reversed by miR-27a-3p agomir (Figure 7A–7D). It thus appears that miR-27a-3p agomir significantly attenuates CI/R responses in vivo.

Figure 6. MiR-27a-3p agomir significantly alleviates the symptoms of CI/R in vivo. (A) Images of brain tissues from rats. (B) Calculated cerebral infraction area. (C) Cerebral water content. (D) TUNEL showing apoptosis within brain tissues. ^**P< 0.01 vs. sham. ^#P<0.05, ^##P<0.01 vs. I/R.

Figure 7. MiR-27a-3p agomir inhibits the expression of FOXO1, p27 Kip1 and active caspase 3 in brain tissues of rats. (A) Protein levels of FOXO1, p27 Kip1 and active caspase 3 in brain tissue from rats. (B–D) Relative expression levels of FOXO1 (B), p27 Kip1 (C) and active caspase 3 (D) normalized to GAPDH levels. ^**P<0.01 vs. sham. ^##P<0.01 vs. I/R.

Cell culture and establishment of in vitro OGD model

HT22 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in an incubator with Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin at 37° C under 5% CO₂/95% O₂. For oxygen-glucose deprivation (OGD) treatment, HT22 cells were placed first in deoxygenated glucose-free medium and then incubated in a hypoxic vessel for 4 h at 37° C under 95% N₂/5% CO₂. The cells were then transferred to DMEM supplemented with high glucose and 10% FBS under normoxic conditions (5% CO₂) at 37° C for 24 h, as previously described [30].

Cell transfection

HT22 cells were cultured in DMEM supplemented with 10% FBS, streptomycin and penicillin (100 U/mL) at 37° C under 5% CO₂. The cells were transfected for 4 h with mimics-control or miR-27a-3p mimics using Lipofectamine 2000. The cells were then washed in warmed medium and incubated for 24 h to establish an in vitro model. Mimics-control and miR-27a-3p mimics were provided with RiboBio (Guangzhou, China).

Quantitative real time polymerase chain reaction (RT-qPCR)

Total RNA was extracted from HT22 cells using TRIzol reagent (Thermo Fisher Scientific) and then reverse transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa, Otsu, Shiga, Japan). RT-qPCR was performed using a SYBR® Premix Ex Taq™ II kit (TaKaRa Bio, Otsu, Shiga, Japan) on a 7900HT system (Applied Biosystems, CA, USA). The PCR protocol entailed incubation at 60° C for 1 min and 90° C for 15 min, followed by 40 cycles at 90° C for 15 s and 55° C for 60 s. The primers used were from GeneCreate Biological Engineering Co., Ltd (Wuhan, China) and were as follows: for MiR-27a-3p, 5’-TCACAGTGGCTAAGTTCCGC-3’ (forward) and 5’-CTCAACTGGTGTCGTGGAGTC-3’. (reverse); for FOXO1, 5’-CAAAATGATGAACCCCAGCTC-3’ (forward) and 5’-CATCCTACCATAGCCATTGCAG-3’ (reverse); for GAPDH: 5’-TGAAGGGTGGAGCCAAAAG-3’ (forward) and 5’-AGTCTTCTGGGTGGCAGTGAT-3’ (reverse) and for U6, 5’-CTCGCTTCGGCAGCACAT-3’ (forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse). The relative mRNA levels were quantified using the 2^−ΔΔCt method and normalized to GAPDH or U6.

Immunofluorescence

HT22 cells were plated onto a 96-well plate at the density of 5.0×10³ cells/well. After incubation, the cells were transfected for 72 h with NC or miR-27a-3p mimics. The cells were then fixed in 4% paraformaldehyde and incubated first with anti-Ki67 antibody (1:100, Abcam Cambridge, MA, USA) overnight at 4° C and then with anti-rabbit IgG secondary antibody (1:1000, Abcam) for 1 h at room temperature. The nuclei was stained with DAPI (Beyotime, Shanghai, China). Finally, the labeled cells were observed under a fluorescence microscope (Olympus BX53 Tokyo, Japan).

CCK-8 assay

HT22 cells (5.0×10³ cells/well) were treated for 48 h with OGD, OGD + mimics-control, or OGD + miR-27a-3p mimics, after which 10 μl of CCK-8 reagents (Beyotime, Shanghai, China) were added into each well, and the plate was incubated for an additional 2 h at 37° C. The absorbance at 450 nm was then measured using a microplate reader (Bio-Rad, Hercules, CA, USA).

Cell apoptosis analysis

After trypsinizing HT22 cells and resuspending them in binding buffer, they were stained with 5 μl annexin V-FITC (BD Biosciences, Franklin Lake, NJ, USA) and propidium iodide (PI; BD Biosciences, Franklin Lake, NJ, USA) for 30 min in the dark at 37° C. Fluorescence-activated cell sorting (FACScan™; BD Biosciences, Franklin Lake, NJ, USA) was then applied to analyze the apoptosis rate using Image J software (BD Biosciences, Franklin Lake, NJ, USA).

ROS detection

HT22 cell suspensions were collected and supplemented with the ROS probe DCFDA (Beyotime, Shanghai, China) as previously described [31]. After 20 min of incubation, cells were centrifuged at 300 g, washed with PBS and resuspended. Relative ROS levels were measured using FACS (FACScan™; BD Biosciences, Franklin Lake, NJ, USA).

Enzyme-linked immunosorbent assay (ELISA)

Supernatants were collected from HT22 cells, and levels of GSH, MDA and SOD were measured using ELISA kits (ELK Biotech, Wuhan, China) according to the manufacturer’s protocols.

Cell cycle detection

HT-22 cells were harvested and counted with a hemocytometer. They were then fixed, permeabilized, stained with 5 μl propidium iodide (PI) and analyzed using FACS with a FACSCalibur (BD Biosciences, Franklin Lake, NJ, USA). The data were quantified using ModFit and FlowJo software.

Western blot analysis

HT22 cells or brain tissues were lysed in RIPA lysis buffer (KeyGEN, Nanjing, China), the protein concentrations in the lysates were determined using a BCA Assay kit (Solar Life Science, Beijing, China). Equal amounts of protein (30 μg) were then subjected to 10% SDS-PAGE, after which the separated proteins were transferred to onto polyvinylidene difluoride membranes (PVDF, Thermo Fisher Scientific). After first blocking the membranes in 5% nonfat dried milk in TBST for 1 h, they were incubated overnight at 4° C with the primary antibodies: anti-active caspase 9 (ab2324, 1:1000), anti-active caspase 3 (ab32042, 1:1000), anti-Bcl-2 (ab182858, 1:1000), anti-FOXO1 (ab179450, 1:1000), anti-p27 Kip1 (ab32034, 1:1000) and anti-GAPDH (ab9485, 1:1000). The membranes were then incubated for 1 h with HRP-labeled goat anti-rabbit secondary antibody (ab7097, 1:5000). Enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) was used to visualize the protein bands. Image J Software was used to quantify the intensity of the bands. All the antibodies were obtained from Abcam.

Luciferase reporter assays

The FOXO1 3ʹ-UTR containing a putative miR-27a-3p binding site (FOXO1 WT 3ʹ-UTR) and a FOXO1 3ʹ-UTR with mutated binding site (FOXO1 MT 3ʹ-UTR) were synthetized by Genepharma (Shanghai, China) and cloned into pmirGLO vectors (Promega, Madison, WI, USA). The vectors (WT or MT) were then transfected into HT22 cells using Lipofectamine 2000 (Thermo Fisher Scientific), and the relative luciferase activity was detected using a dual luciferase reporter kit (Promega).

In vivo experiment

Six-week-old Wistar rats were purchased from Vital River (Beijing, China) and housed within a dedicated specific pathogen-free facility. The rats were randomly divided into three groups: (1) sham-operated (Sham) (n=12); (2) I/R (n=12); and (3) I/R + miR-27a-3p agomir (n=12). To mimic CI/R in vivo, rats in groups (2) and (3) were anesthetized with 45 mg/kg sodium pentobarbital, and the middle cerebral artery (MCA) was occluded with a surgical filament. After 1 h of MCA occlusion, the filament was removed, and reperfusion proceeded. The rats in group (1) underwent the same procedure except for the filament insertion. Rats in groups (1) and (2) were injected with saline via intraventricular. Rats in group (3) were injected with miR-27a-3p agomir (RioboBio, Guangzhou, China) via intraventricular. At the end of the study, rats were sacrificed for the collection of brain tissues. Meanwhile, the apoptotic cells were detected by TUNEL staining as previously described [32].

At the end of the study, the rats were sacrificed, and the brain tissues were collected. Cerebral infarction area and cerebral water content were assessed. All in vivo experiments were performed in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals, following a protocol approved by the Ethics Committees of Zhejiang University School of Medicine.

2, 3, 5-Triphenyltetrazolium chloride (TTC) staining

In brief, the brains tissues from different groups were collected and frozen for 30 min at - 20° C. Then, the brain tissues were sliced into 2-mm-thick sections and incubated with 2% TTC solution (Sigma, St. Louis, MO, USA) at 37° C for 20 min, which was terminated by rinsing with PBS. Subsequently, the slice section was fixed with 4% paraformaldehyde for 2 h and photographed. The cerebral infarction area was expressed as a percentage of total infarct volume/total brain volume × 100%.

Statistical analysis

All data are expressed as the mean ± standard error (S. D.). CCK-8 assays, immunofluorescence staining, western blotting, RT-qPCR, ELISAs, flow cytometry and ROS detection were performed in triplicate. In addition, all experiments were repeated three times. One-way analysis of variance (ANOVA) and post hoc Tukey’s tests were used for comparisons between at least three groups. Values of P<0.05 were considered statistically significant.

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All in vivo experiments were performed in accordance with National Institutes of Health guide for the care and use of laboratory animals, following a protocol approved by the Ethics Committees of Zhejiang University School of Medicine.