MicroRNA-194 Inhibition Promotes SUMO-2-Dependent Neuro-Protection Against Oxygen-Glucose Deprivation

Hypothermia is a powerful neuroprotectant. However, clinical translation has been dicult partly because the underlying mechanisms remain to be fully elucidated. Recently, it has been suggested that hypothermic neuroprotection may be linked with specic microRNA signatures, specically the downregulation of miR-194-5p. Here, we attempt a reverse translation study to dene the novel neuroprotective mechanism of miR-194-5p downregulation. Rat brain endothelial cells; PI: Propidium Iodide Incorporation; TS: targeting sequence; WTS: with wide targeting sequence; MTS: with mutated targeting sequence; MOI: multiplicity of infection; SUMO-2: small ubiquitin-related modier-2; UTR : untranslated region.


Results
First, we documented that miR-194-5p was highly expressed in rat primary cortical neurons and astrocytes, compared with other glial and vascular cell types. Blockade with anti-miR-194-5p did not affect astrocytes, but signi cantly protected neurons against oxy-glucose deprivation. Using a miRNA target prediction algorithm, we found that SUMO-2, a known endogenous neuroprotective regulator, possessed the binding site for miR-194-5p. When miR-194-5p was inhibited, SUMO-2 mRNA was increased in neurons along with the enhancement of SUMO-2 conjugation. Finally, downregulation of SUMO-2 with none-special puromycin and special lentiviral shRNA-mediated knockdown of SUMO2 both canceled the neuroprotection mediated by miR194-5p inhibition.

Conclusion
Taken together, this study provides the rst proof-of-concept that miR194-5p may be a negative controller of endogenous SUMO-2 protective mechanisms, and therefore miR194-5p inhibition may provide a novel approach for leveraging hypothermic neuroprotection against ischemic stress.
Background Page 3/15 Many neuroprotective treatments are effective in cell and animal models. However, the translation of experimental studies to humans has not been broadly successful. Within the wide spectrum of neuroprotective approaches that have been assessed to date, hypothermia appears to be one of the most e cient treatments for protecting brain, including resuscitation after cardiac arrest, complex cardiovascular surgery, and protection against cerebral ischemia (1)(2)(3). Although the potential of hypothermia to protect human brain is well-accepted, little is known about the underlying mechanisms.
Thus, understanding the fundamental mechanism of hypothermic neuroprotection will enable this approach to be more widely and effectively applied.
In our previous study using a piglet model of deep hypothermic circulatory arrest, we identi ed signi cant microRNA signatures in the hippocampus CA1 region, speci cally the downregulation of miR-194-5p as the response with the highest q value and biggest fold-change (4), suggesting that miR-194-5p may be responsible for the mechanisms of hypothermia. In this study, we attempted a reverse translation study to de ne the role of miR-194-5p in neuroprotection against oxygen-glucose deprivation (OGD). Our ndings showed that inhibition of miR-194-5p promotes neuronal survival via the enhancement of endogenous neuroprotective machinery involving SUMO-2 activation.

Cell cultures
Rat primary cell cultures: Primary cultures for rat cortical neurons, rat cortical astrocytes, and rat cortical microglia were prepared as described previously (20)(21)(22). All cells were cultured at 37 °C in a humidi ed chamber of 95% air and 5% CO 2 . Primary neuron cultures. Primary neuron cultures were prepared from cerebral cortices of embryonic day (E)17 Sprague-Dawley rat embryos. In brief, cortices were dissected and dissociated using papain dissociation system (Worthington Biochemical Corporation, LK003150).
Primary neuron were spread on plates coated with poly-d-lysine (Sigma, P7886) and cultured in DMEM (NBM, Life Technology, 11965-084) containing 25 mM glucose, 4 mM glutamine, 1 mM sodium pyruvate, and 5% FBS at a density of 2 × 10 5 cells ml−1 (1 ml for 12-well format, 0.5 ml for 24-well format). At 24 h after seeding, the medium was changed to Neurobasal medium (Invitrogen, 21103-049) supplemented with B-27 (Invitrogen, 17504044) and 0.5 mM glutamine. Primary neurons were cultured at 37 °C in a humidi ed chamber of 95% air and 5% CO2. Primary neuron cultures were used for experiments from 7 to 10 days after seeding. Rat cortical astrocytes Primary astrocyte cultures were prepared from cerebral cortices of 2-day-old neonatal Sprague-Dawley rats. In brief, dissociated cortical cells were suspended in DMEM (Life Technology, 11965-084) containing 25mM glucose, 4mM glutamine, 1mM sodium pyruvate, and 10% FBS and plated on uncoated 25cm2 asks at a density of 6×105 cells cm − 2. Monolayers of type 1 astrocytes were obtained 12-14 days after plating. Non-astrocytic cells such as microglia and neurons were detached from the asks by shaking and removed by changing the medium. The microglia were collected for further culture. Astrocytes were dissociated by trypsinization and then reseeded on uncoated T75 asks. After the astrocytes reached 70-80% con uence, were used for experiments. Rat brain endothelial cells: A rat brain microendothelial cell line, RBE.4 were maintained in EBM-2 containing EGM-2MV Single Quots kit onto collagen-coated 25 cm 2 asks at a density of 2×10 5 cells/cm 2 incubated in a 5% CO 2 incubator at 37°C.

RNA extraction and real-time PCR.
Total RNA was extracted from the primary cultured cells using RNeasy Plus Mini Kit (50) (QIAGEN, Cat. No. 74134) in accordance with the manufacturer's instructions. Total RNA was reverse-transcribed with hairpin-loop primers designed to target the speci c miRNA at a concentration of 600 ng/μL used for cDNA synthesis PrimeScript TM 1 st strand cDNA Synthesis Kit (TaKaRa Clontech, Cat#6110A). Real-Time Quantitative Reverse-Transcription Polymerase Chain Reaction was performed with 20 ng cDNA in a 20 μL volume on the ABI 7500 System. Semi-quantitative real-time PCR, using RT 2 SYBR Green ROX qPCR Mastermix (QIAGEN, Cat. No. 330520), For each of the selected miRNAs, real-time PCR measurements were performed to obtain a mean CT value for each sample. The CT values of the different samples were compared using the 2 -ΔΔCT method, and U6 expression levels were used as an internal reference. For mRNA assays total RNA was reverse-transcribed with oligoDT primers at a concentration of 600 ng/μL used for cDNA synthesis PrimeScript TM 1 st strand cDNA Synthesis Kit (TaKaRa Clontech, Cat#6110A). TaqMan mRNA assays (Applied Biosystems Inc., Carlsbad, CA, USA) were used to quantify SUMO-2 mRNA (Rn00821719-g1, invitrogen) expression levels, in accordance with the manufacturer's protocol. and B2m (Rn00560865-m1, invitrogen) expression levels were used as an internal reference.
Oxygen-glucose deprivation (OGD) and reoxygenation OGD experiments were performed using a specialized, humidi ed chamber (Heidolph, incubator 1000, Brinkmann Instruments) kept at 37 °C, which contained an anaerobic gas mixture (90% N2, 5% H2, and 5% CO2) as we descripted before (22). To initiate OGD, culture medium was replaced with deoxygenated, glucose-free DMEM (Life Technology, 11966-025). After 2 h challenge, cultures were removed from the anaerobic chamber, and the OGD solution in the cultures was replaced with maintenance medium. Cells were then allowed to recover for 18 h (for neurotoxicity assay) in a regular incubator.

Determination of cell viability
Neuronal injury was measured by standard cell cytotoxicity assays such as Cell Counting Kit 8 cytotoxicity assay (DOJINDO, CK04-13) and PI (Propidium Iodide Incorporation) staining. Cell viability was quanti ed using a Cell Counting kit-8 (CCK-8, Dojindo) according to the manufacturer's instructions. CCK-8 solution (10 μl) was added to each well of the plate, and the cells were incubated at 37 C for 2 hours. The optical density at a wavelength of 450 nm was measured with microplate reader. The relative assessments of neuronal injury were normalized by comparison with control cell as 100% cell survival (CCK8). For the PI staining, Cell viability was assessed after staining of naive cell cultures with propidium iodide (PI) to distinguish between living and dead cells (0.001mg/mL for 10minutes with subsequent rinsing) and xed with 4% paraformaldehyde in phosphate buffered saline. Five images per well were taken using microscope. Viable neurons not incorporating PI were counted in transmission images and quanti ed.
Luciferase reporter assays were performed to verify the direct binding of miR-194-5p to SUMO2 transcripts.
Construction of 3′-UTR reporter plasmids and luciferase assays synthesized double-strand oligonucleotides containing 20 bases upstream and 15 bases downstream of miR-194-5p seed sequences. We constructed pmiR-GLO-194-5p SUMO2-TS (targeting sequence) reporter vector, containing the 3′-UTR of SUMO2. The pmiRGLO-194-5p-SUMO2-WTS (with wide targeting sequence) also generated in this cloning step. The pmiRGLO-194-5p-SUMO2-MTS (with mutated targeting sequence) also generated in this cloning step, by reversing the seed targeting sequence of SUMO2. The 293T cells were cotransfected with either pmiRGLO-194-5p-SUMO2-MTS or pmiRGLO-194-5p-SUMO2-WTS with miR-Con or miR-194. Luciferase reporter assay cells were lysed in passive lysing buffer and then analyzed for the re y and renilla luciferase activities using the commercial Dual-Luciferase reporter assay system on the GloMax-multi Detection Luminometer (Promega Corporation, E7031, Madison, WI, USA), and the re y luciferase activity was normalized to the renilla luciferase activity. In all, 24 h after transfection, the re y and Renilla luciferase activities were detected consecutively using Dual-Luciferase Kit (Promega Corporation). Relative protein levels were expressed as Fire y/Renilla luciferase ratios.  Fig. B). Image acquisition was executed using an inverted uorescence microscope (Olympus) equipped with a 60× oil immersion objective. Sustained SUMO2 downregulation levels were con rmed by western blot analysis neurons after transfection ( Supplementary Fig. C).

Statistical analysis.
All values are represented as mean± SD of at least three independent experiments. When only two groups were compared, Student's t-test was used. Multiple comparisons were evaluated by one-way ANOVA followed by Tukey's tests. P-values of <0.05 were deemed to be signi cantly different.

Discussion
Taken together, these data suggest a novel mechanism that miR-194-5p inhibitor may up-regulated SUMO-2, thus blocking miR-194-5p may be a potential therapy for protecting neurons against ischemic stress. microRNAs (miRNAs) are short, non-coding RNAs that bind to mRNAs to inhibit translation and/or promote mRNA degradation by imperfect base-pairing between the seed region in miRNAs (7). Emerging evidence suggest that miRNAs play important roles in CNS. For example, miR-124, the most abundant miRNA in the CNS, regulates neuroprotection by preventing neuronal apoptosis (8) and modulating in ammatory response through inducing anti-in ammatory phenotype in microglia/macrophage (9) after cerebral ischemia. More recently, it has been reported that hypothermia may alter miRNA response after traumatic brain injury, suggesting the presence of temperature-sensitive miRNAs in the brain (10). From our previous study using a piglet model of deep hypothermic circulatory arrest, we identi ed miR-194-5p as a leading candidate for hypothermia-sensitive miRNA and neuroprotection (4). Here, we demonstrated the miR-194-5p may speci cally recognize SUMO-2 mRNA and downregulate gene expression by translational repression or mRNA cleavage posttranscriptional mechanisms.
SUMO-2/3 conjugation is indeed involved in the protective effects induced by deep hypothermia and may play a role in protein quality control such as subcellular localization, transcription regulation, DNA replication, and repair (11)(12)(13). SUMO-2 conjugation and levels of SUMO-conjugated proteins markedly increase in hibernating animal brain during the torpor phase. Therefore, it has been suggested that this may be a protective response shielding neurons from damage induced by low blood ow and substrate deprivation (14). Moreover, accumulating studies veri ed that global increases in SUMO-2/3 and simultaneous SUMOylation are endogenous neuroprotective response to ischemic stress (15)(16)(17). Our current study showed that SUMO-2 conjugation was signi cantly increased when miR-194-5p was blocked in neurons, suggesting that inhibition of miR-194-5p-SUMO-2 interaction may be a key mechanism in hypothermia-mediated brain protection.
Nevertheless, there are several caveats that should be considered. Our data implicate miR-194-5p as a candidate for hypothermia-sensitive miRNA. But other miRNAs such as miR-874 and miR-451 are also known to be temperature sensitive (10). How miR-194-5p interacts with other miRNA networks remain to be fully dissected. A second caveat involves the role of miR-194-5p in other brain cells. We found that miR-194-5p was also highly expressed in astrocytes. Many miRNAs have been described to explain astrocytic functional activation including astrogliosis, induction of in ammation, and enhancement of neuroplasticity (18). Although miR-194-5p inhibitor did not affect cell survival or proliferation in astrocytes following ischemic stress, miR-194-5p may still affect neuron-glia crosstalk in other ways. The miR-194-5p-SUMO-2 interaction occurs in ischemic brain should be eventually addressed with in vivo studies. Finally, based on our previous our ndings that hypothermia inhibited miR-194-5p expression, we tested the effect of miR-194-5p inhibition on neuroprotection. Of course, hypothermia inhibits many different pathways including excitotoxicity, neuroin ammation, apoptosis and free radical production (19). Further studies are required to de ne how miR-194-5p inhibition may affect these pathways.

Figure 2
SUMO-2 may be a target of miR-194-5p in rat cortical neurons. A. In miRNA target prediction algorithm, we found that SUMO-2 had the binding site for miR-194-5p within its 3`-untranslated region (UTR) in most species including human. B. qRT-PCR analysis demonstrated that SUMO-2 mRNA was signi cantly increased by miR-194-5p inhibition (n=3). C. Western blot showed that accumulation of SUMO-2 conjugates were clearly increased by the inhibition (n=5). D. Using luciferase assays we determine miR-194 can directly target the 3'UTR of SUMO-2. miR-194 can bind to the SUMO-2 3'UTR to regulate gene expression.