MicroRNA‐410 serves as a candidate biomarker in hypoxic‐ischemic encephalopathy newborns and provides neuroprotection in oxygen‐glucose deprivation‐injured PC12 and SH‐SY5Y cells

Abstract Background MicroRNA‐410 (miR‐410) has been found to be deregulated in neonatal hypoxic‐ischemic encephalopathy (HIE). However, the clinical significance and biological function of miR‐410 remain largely elusive. This study aims to investigate the expression and diagnostic performance of miR‐410 in HIE newborns, and explores the neuroprotective effect of miR‐410 in an oxygen‐glucose deprivation (OGD)‐induced cell injury model. Methods Expression of miR‐410 was examined using quantitative real‐time PCR, and its diagnostic performance was evaluated using a receiver operating characteristic analysis. We used OGD‐injured PC12 and SH‐SY5Y cells to construct an in vitro HIE model. The effect of miR‐410 on OGD‐induced cell injury was analyzed by assessing cell viability and apoptosis. Enzyme‐linked immunosorbent assay was used to evaluate inflammation in cell model. A target gene was assessed according to the luciferase reporter assay. Results Serum miR‐410 expression was significantly decreased in HIE newborns and OGD‐injured cell model. The reduced miR‐410 expression served as a biomarker for the diagnosis and progression of HIE. The OGD‐induced impaired cell viability, enhanced cell apoptosis, and activated neuroinflammation were abrogated by the overexpression of miR‐140 in both PC12 and SH‐SY5S cells. Regarding the mechanisms underlying the function of miR‐410, phosphatase and tensin homolog (PTEN) was proposed as a direct target of miR‐410. Conclusion All data revealed that serum downregulated miR‐410 in HIE serves as candidate diagnostic biomarker, and that miR‐410 exerts a neuroprotective role in OGD‐injured cells by improving cell viability and inhibiting cell apoptosis through targeting PTEN.


INTRODUCTION
Neonatal hypoxic-ischemic encephalopathy (HIE) is a disorder with hypoxic and ischemic brain damages and mainly caused by perinatal asphyxia (Laptook, 2016). It is associated with high neonatal mortality and leads to long-term neurologic morbidity (Glass, 2018). In developed countries, the incidence of HIE accounts for 1-8 per 1000 live newborns, and this data grow to 26 per 1000 in underdeveloped counties (Liu et al., 2017). To date, the therapeutic hypothermia is the only recognized treatment for neonatal HIE, but this strategy needs to be administrated within 6 h after birth, and its application in severe HIE cases is not ideal (Chiang et al., 2017). Thus, early diagnosis and efficient therapeutic approaches are urgently needed for newborns suffering from HIE.
MicroRNAs (miRNAs) are a series of small noncoding RNAs with important regulatory effects on a wide variety of biological processes, such as cell proliferation, migration, invasion, differentiation and apoptosis (Chao et al., 2018). Emerging evidence shows that human diseases are generally accompanied with dysregulation of miRNAs (Vishnoi and Rani, 2017). These aberrant miRNAs can serve as biomarkers for disease diagnosis and progression and participates disease pathogenesis (Bertoli et al., 2015). Some functional miRNAs have also been identified in neonatal HIE with pivotal roles in HIE development and progression, including miR-210 (Ma et al., 2016), miR-17-5p , and miR-204 (Chen et al., 2019). A recent study by O'Sullivan has investigated the altered miRNAs expression in umbilical cord blood in neonatal HIE, and microRNA-410 (miR-410) is one of the deregulated miRNAs in whole blood samples (O'Sullivan et al., 2019). In addition, miR-410 has been documented to serve a protective role against brain injury in Parkinson's disease (Ge et al., 2019), anesthesia-induced cognitive dysfunction (Su et al., 2019) and ischemic stroke . However, the clinical significance and biological function of miR-410 in neonatal HIE remain largely elusive.
To improve the diagnosis of neonatal HIE, this study sought to evaluate the expression and diagnostic performance of miR-410 in HIE newborns. In addition, this study constructed an oxygen and glucose deprivation (OGD)-induced cell injury model in PC12 and SH-SY5Y cells as the most commonly applied in vitro model for HIE  to examine the biological function of miR-410 in regulating neuronal cell survival.

Study subjects and serum sample collection
One hundred and two HIE newborns were included in this study, who were collected from Weifang People's Hospital between May 2015 and April 2018. All the HIE newborns were full term infants without any brain injury caused by hyperbilirubinemia, intrauterine infection or severe brain dysplasia, and did not receive any therapy before sampling. The diagnosis of HIE was performed in accordance with the published criteria by Sarnat et al. (Sarnat and Sarnat, 1976) and confirmed with brain MRI results. In addition, 60 healthy newborns were collected as controls during a same time period, and none of them had medical history of perinatal asphyxia or disorder in nervous system after birth. According to the criteria by the Group of Neonatology et al. (Group of et al., 2005), the enrolled HIE newborns were grouped into mild, moderate and severe groups to facilitate further analyses.
Femoral vein blood was collected from the newborns within 6 h after birth, then was centrifuged for the extraction of serum samples, which were stored at −80 • C for further analyses. The protocols of this study were approved by the Ethics Committee of Weifang People's Hospital, and a written informed consent was obtained from each newborn's parent.

Construction of oxygen-glucose deprivation-induced cell injury model
OGD experiment has been widely used to induce neuronal cell injury as an in vitro HIE model. In this study, PC12 and SH-SY5Y cells were used and firstly cultured in glucose-free medium at 37 • C in a hypoxia incubator with 5% CO 2 , 94% N 2 , and 1% O 2 for 2 h. Thereafter, the culture medium was replaced with growth medium containing glucose and incubated at 37 • C under a normal condition with 5% CO 2 . The cells cultured under normal condition (37 • C in a humidified incubator with 5% CO 2 ) with DMEM supplemented 10% FBS were used as controls. For the cells with artificially regulated miR-410, the OGD induction was conducted at 24 h after cell transfection.

RNA extraction and quantitative real-time PCR
Total RNA in serum and cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA were synthesized from RNA by a PrimeScript RT reagent kit (TaKaRa, Shiga, Japan) as per the manufacturers' instruction. Quantitative real-time PCR (qRT-PCR) was carried out to evaluate mRNA expression with the SYBR green I Master Mix kit (Invitrogen, Carlsbad, CA, USA) on a 7500 real-time PCR system (Applied Biosystems, USA). The final relative expression value was calculated using the 2 −ΔΔCt method and normalized to cel-miR-39-3p or GAPDH.

Cell viability analysis
Cell viability of PC12 and SH-SY5Y was evaluated using MTT assay.
Briefly, cells with a density of 5 × 10 3 cell per well were seeded into 96-well plates and cultured at 37 • C with 5% CO 2 for 24 h, then 0.5 mg mL −1 MTT solution was added into the cells for 4 h. After remove the MTT solution (Sigma-Aldrich, MO, USA), 100 μL DMSO was added into the cells to dissolve the violet formazan crystals. The absorbance at 570 nm was measured to reflect the viability of PC12 and SH-SY5Y cells.

Cell apoptosis analysis
Cell apoptosis was examined using flow cytometry analysis. Cells were

Enzyme-linked immunosorbent assay
To evaluate the inflammatory responses in the cell model, proinflammatory cytokine levels were analyzed. Enzyme-linked immunosorbent assay kits (R&D System, Abingdon, UK) were used to measure the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α following the manufacturer's protocols.

Dual-luciferase reporter assay
The miRanda bioinformatics software (www.microrna.org/microrna/ home.do) was used to seek the putative target genes for miR-410, and phosphatase and tensin homolog (PTEN) was found to possess the binding site of miR-410 at its 3'-UTR. A dual-luciferase reporter assay was carried out to check the relationship between miR-410 and PTEN.
The wild-type (WT) and mutant-type (MT) 3'-UTR of PTEN were separately cloned into the pmirGLO luciferase reporter vectors (Promega), and the combined vectors were co-transfected into SH-SY5Y cells with miR-410 mimic or mimic NC using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 48 h of transfection, the relative luciferase of each group was measured using the Dual-luciferase reporter assay system (Promega).

Statistical analysis
All the statistical analyses were performed with SPSS 21.0 software (SPSS Inc., Chicago, IL) and GraphPad Prism 7.0 software (GraphPad Software, Inc., USA). Quantitative data were presented as mean ± SD. Differences between groups were analyzed using Student's t-test and one-way ANOVA followed by Tukey's test. The receiver operating characteristic (ROC) curves were plotted to evaluate the clinical significance of miR-410 in HIE newborns, and the area under the curve (AUC) was calculated to reflect the diagnostic accuracy of miR-410. The results with P < .05 were considered statistically significant.

General characteristics of study newborns
The general characteristics of the enrolled newborns were recorded and listed in

Expression of microRNA-410 in hypoxic-ischemic encephalopathy newborns
Serum levels of miR-410 were examined using qRT-PCR. The analysis results shown in Figure 1(A) indicated that HIE newborns had lower serum miR-410 expression than the healthy controls (P < .01). By comparing miR-410 expression in HIE newborns with different severity, the highest expression was observed in mild HIE group, followed by the moderate group, and the lowest expression was presented in serve HIE group (P < .01, Figure 1(B)).

Clinical significance of microRNA-410 in neonatal hypoxic-ischemic encephalopathy diagnosis
Given the deregulated expression of miR-410 in HIE newborns, its diagnostic performance was evaluated using a ROC curve. As shown in Figure 2

MicroRNA-410 significantly ameliorates oxygen-glucose deprivation-induced cell injury and inflammatory responses
The neuronal cell injury was measured by analyzing cell viability and apoptosis. As shown in Figure 3

Phosphatase and tensin homolog is directly regulated by microRNA-410
By bioinformatics analysis, PTEN was predicted as a potential target gene of miR-410, and the alignment of the seed regions of miR-410 with the 3'-UTE of PTEN was shown in Figure 4

DISCUSSION
Emerging studies have demonstrated that miRNAs serve important roles in the development and progression of brain injury-related diseases (Sarkar et al., 2019). For example, Xi et al. have reported that intracerebral hemorrhage patients had reduced expression of miR-27a-3p, and the overexpression of miR-27a-3p could significantly attenuate blood-brain barrier disruption and brain injury (Xi et al., 2018). The increased levels of miR-124-3p in microglial exosomes have been documented to be involved in the progression of traumatic brain injury through regulating inflammation and neuronal cell as a potential therapeutic target for ischemic stroke by modulating neuronal cell apoptosis (Shi et al., 2020). These aforementioned studies indicate that brain injury-associated diseases are generally accompanied with aberrant miRNAs, which may act pivotal roles in disease pathogenesis.
In neonatal HIE, there are also some functional miRNAs. A study by  was elevated in neonatal HIE rats and the inhibition of miR-200b-3p had a beneficial effect on the brain damage . The upregulated expression of miR-210 has also been reported in neonatal HIE and was considered as a therapeutic target by regulating microglia-mediated neuroinflammation . participate the development of brain injury in some diseases (Ge et al., 2019, Su et al., 2019. Thus, we suspected that miR-410 might serve a potential role in the development and progression of neonatal HIE. Considering the aberrant expression of miR-410 in serum samples of HIE newborns, its clinical significance was further evaluated. miR-NAs are considered a group of good diagnostic tools for various human diseases, owing to their stability and detectability in serum samples (Hayes et al., 2014). Some miRNAs with deregulated expression levels have been proposed as biomarkers for neonatal HIE diagnosis or prognosis, such as miR-210 and miR-374a . In this study, the diagnostic value of serum miR-410 was evaluate using ROC analysis, and the results suggested that serum miR-410 had relatively high diagnostic accuracy for screening HIE newborns from healthy controls. In addition, given the significant differences in miR-410 levels between HIE neonates with different severity, the discriminating potential of miR-410 for neonatal HIE severity was investigated.
The ROC curves indicated that miR-410 could be used as biomarker to predict the severity of neonatal HIE. However, the sample size in our study was finite, which might limit the result accuracy. Thus, further investigations are necessary to confirm our conclusion with a large-scale study population.
To This study predicted the putative target genes of miR-410, and analyzed the relationship of PTEN, which was one of the predicted targets with important role in neuroprotection, with miR-410 in the OGD-induced cell injury model. PTEN is an important upstream regulator of the AKT signaling pathway and its inhibition can prevent neuron injury after hypoxia-ischemia (Zhao et al., 2013). In neonatal HIE, PTEN has been documented to mediate the protective effect of sevoflurane post-conditioning against brain injury (Xue et al., 2019).
In addition, the neuroprotective role of miR-410 has been reported in Parkinson's disease by targeting PTEN (Ge et al., 2019). In this study, an increase in the mRNA expression of PTEN was obtained in the OGD-induced cell injury model, and this effect was significantly attenuated by miR-410, which inspired us that miR-410 in neonatal HIE might also exert neuroprotection by targeting PTEN. However, the molecular mechanisms underlying the function of miR-410 need to be further explored regarding the related signaling pathways. In addition, another limitation of this study is the lack of in vivo animal experiments to confirm the mechanisms of miR-410. Thus, further explorations using neonatal HIE animal models are necessary to further uncover the role and mechanisms of miR-410 in neonatal HIE.
Taken together, our analysis results reveal that serum reduced miR-

CONFLICTS OF INTEREST
The authors have declared no conflict of interest.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are available from the corresponding author upon reasonable request.