Highly expressed miR-210 in the peripheral blood is closely associated with asphyxiated neonates

Introduction

In China, neonatal brain injury caused by asphyxia is one of the most common reasons for neonatal disability and death. Asphyxia has high rates of morbidity and disability, with an unclear mechanism (1). Traditional indicators and clinical symptoms have limited specificity for diagnosing neonatal brain injury caused by asphyxia, and subjective evaluations are primarily used in clinical settings; however, they also have limited sensitivity and specificity, insufficient stability, and often lag in terms of pathophysiological changes (2). Each individual’s tolerance to hypoxia varies greatly, making it difficult to diagnose and prognostically predict neonatal brain injury caused by asphyxia early. Neonatal brain injury caused by asphyxia is an issue tormenting the field of pediatrics. Therefore, it is crucial to untimely identify asphyxial hypoxia before the initiation of brain injury by using an early targeted treatment to block the injury.

MicroRNAs (miRNAs) are a class of noncoding small-molecule RNAs that can rapidly and efficiently degrade target messenger RNAs (mRNAs) or inhibit protein translation by binding to target sites (3,4). miRNAs play a role in almost all physiological and pathological processes and can act as early predictive molecular markers of diseases (5-7). A study revealed that when the brain tissue is damaged, brain miRNAs are altered and secreted into the peripheral blood circulation (8). Therefore, miRNAs can serve as diagnostic markers of brain tissue injury (9). Furthermore, related animal experiments have suggested that miR-210 expression is downregulated in the brain tissue of model rats and that it participates in the process of brain injury (10). To date, however, only a few clinical studies have reported the role of miR-210 in the diagnosis of brain injury in neonates with early asphyxia.

In the present study, we determined the changes in miR-210 expression in the peripheral blood of neonates with asphyxia and analyzed the correlation between miR-210 and clinical indicators of brain injury in early asphyxia. Furthermore, we elucidated the clinical characteristics of its upregulation in predicting brain injury prognosis. Moreover, we identified the miR-210 target gene-related pathways and diseases and analyzed the network interactions among the miR-210 target genes associated with neurological and cardiovascular diseases. We present this article in accordance with the STARD reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-23-106/rc).

Methods

Patient information

The study subjects were 27 neonates with asphyxia (gestational age >37 weeks) who had been admitted to the Neonatology Department of The Third People’s Hospital of Dongguan between January 2015 and December 2016. The control group comprised 26 normal neonates who were recruited during the same period. The patients in the asphyxia group have a gestational age of 37–42 (40.62±0.71) weeks and weighed 2.6–4.1 (3.55±0.42) kg. The patients in the control group had a gestational age of 37–42 (40.62±0.74) weeks and weighed 2.6–4.4 (3.59±0.45) kg. The neonates in the control group had no diseases. A difference test was conducted to determine the comparability of the groups for analysis. All the study participants’ parents signed an informed consent form consenting to their child’s inclusion in the study. The study was conducted according to the Declaration of Helsinki (as revised in 2013) and approved by the Ethics Committee of The Third People’s Hospital of Dongguan (No. 2022-KYSB-014). None of the selected patients were treated for mild hypothermia.

The inclusion criteria were formulated according to the diagnostic criteria for neonatal asphyxia jointly formulated by the American Academy of Pediatrics and the College of Obstetrics and Gynecology in 1996 and were as follows: neonates (I) with severe metabolic/mixed acidosis (pH <7.00) in the umbilical artery blood; (II) with an Apgar score of 0–3 points and duration of >5 min; (III) with early neurological manifestations, such as convulsions, coma, or hypotonia; and (IV) with evidence of multiple organ dysfunction during premature delivery. The patients who met the above criteria were diagnosed with asphyxia. Neonates who met the abovementioned criteria and had multiorgan (≥3 organs) damage and/or hypoxic–ischemic encephalopathy were diagnosed with severe asphyxia.

The exclusion criteria were as follows: (I) neonates with other serious diseases that may have affected the comparability of the present study, such as a congenital genetic metabolic disease, neonatal hemorrhage, neonatal hemolytic disease, septic shock, and birth trauma owing to intracranial hemorrhage, and/or (II) those who were diagnosed with hypoxic-ischemic encephalopathy (HIE) and were excluded after 2 weeks of treatment and observation as well as based on laboratory results and head magnetic resonance imaging (MRI).

HIE was diagnosed based on diagnostic criteria, clinical symptoms, Apgar score, and potential of hydrogen (pH) of umbilical cord blood at delivery. Figure 1 presents the study flow chart.

Figure 1 A flow chart depicting the study protocol. NBNA, neonatal behavioral neurological assessment; MRI, magnetic resonance imaging; qRT-PCR, quantitative real-time polymerase chain reaction.

Quantitative real-time polymerase chain reaction

Peripheral blood (1 mL) was collected from the patients in each group within 1 h of their birth. According to the gene sequences, Primer Premier 5 (Premier, Canada) was used to design the primers. Next, total RNA and miRNA were extracted using the TRIzol kit or HiPure Blood/Liquid RNA Kit (Magen, R4163) according to the manufacturer’s instructions. RNA quality was determined using an ultra-micro ultraviolet spectrophotometer to determine RNA concentration and purity. For RNA purity detection, an A260/A280 ratio of 1.8–2.1 indicated that the purity was qualified. Agarose gel electrophoresis was performed to determine the quality of total RNA in the sample. RNA was dissolved in RNA kinase-free water and stored at −80 ℃. Complementary DNA reverse transcription was performed using M-MLV Reverse Transcriptase (Promega, M1705) using the following experimental system: 2 µg of RNA, 1 µL of Primer Mix, and RNase-free water to 15 µL; 5 min at 70 ℃ and 5 min on ice; 5×5 µL of buffer, 6.25 µL of 2 mM dNTP, 1 µL of M-MLV, and 12.75 µL of RNase-free water, for a total reaction mix of 25 µL; and 60 min at 42 ℃. Next, amplification was performed using the ChamQ SYBR qPCR Master Mix (Vazyme, Q341-03). The following formula was used to calculate the relative expression of the target gene: 2^−ΔΔCt = 2 − [(ΔCt) Test − (ΔCt) Control].

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses

The DAVID database (https://david.ncifcrf.gov/tools.jsp) was used to determine the targeted genes via classical GO and KEGG pathway enrichment analyses. The data were processed using the cluster Profiler package in R (version 3.14.3) and the org.Hs.eg.db package (version 3.10.0). GO analysis was performed to elucidate the biological processes (BPs) of the genes, which can be used to predict protein function. Furthermore, KEGG pathway analysis was conducted to assign the differentially expressed genes to specific pathways to identify the metabolic pathways that were significantly altered in the disease states.

Construction of the protein-protein interaction (PPI) network

First, the miR-210 target genes involved in neurological or cardiovascular diseases were imputed into the STRING database (https://string-db.org/); the combined score was set to >0.4. Next, the calculation results were imported into Cytoscape-v3.8.2 to construct the PPI network. The line between two points in the PPI network diagram represents the relationship between two genes with direct effects.

Statistical analysis

Statistical analysis was performed using SPSS 21.0 software (SPSS, Armonk, NY, USA). The normally distributed measurement data were presented as mean and standard deviation (x¯ ± standard deviation). Differences between the groups were compared using the t-test. The non-normally distributed measurement data were presented as a median and interquartile range [M (Q)]. The rank-sum test was used to compare the differences between the groups. The count data were presented as the number of cases and percentage [n (%)]. The exact probability method was used to compare the differences between the groups. A P value of <0.05 was considered statistically significant.

Results

miR-210 was highly expressed in the peripheral blood of neonates with asphyxia

We observed that miR-210 expression was significantly upregulated in the asphyxia group compared with the control group (Figure 2A and Table 1). However, we observed that the number of normal deliveries, cord pH, and 1-, 5-, and 10-min Apgar scores were markedly decreased in the asphyxia group compared with the control group (Table 1). We also elucidated the relationship between miR-210 expression and pathological indicators and observed that miR-210 expression was not related to 1-, 5-, and 10-min Apgar scores in the HIE group; the possible reason for this may be the insufficient sample size of our study (Figure 2B).

Figure 2 The expression of miR-210 in the peripheral blood of asphyxiated neonates and its correlation to the Apgar score. (A) The expression of miR-210 in the peripheral blood of the neonates was tested by qRT-PCR for the control (n=26) and asphyxia groups (n=27). (B) Correlation analysis of miR-210 expression and the 1-, 5-, and 10-min Apgar scores. *, P<0.05; **, P<0.01. HIE, hypoxic-ischemic encephalopathy; qRT-PCR, quantitative real-time polymerase chain reaction.

The relationship between miR-210 expression and the clinical indicators

The incidence of coma, low muscle tension, and respiratory distress in the miR-210 high-expression group were higher than those in the miR-210 low-expression group (Table 2), and the difference was statistically significant. However, no significant differences were found in white blood cell (WBC) numbers, procalcitonin (PCT) levels, pH, neonatal behavioral neurological assessment (NBNA) abnormalities, abnormal imaging examinations, clinical irritation, and high muscle tension between the two groups.

Relationship between miR-210 expression and the indicators of neonatal asphyxia

Differences in miR-210 expression between the normal and abnormal NBNA groups, normal and abnormal low-pH groups, and normal image and abnormal impact examination groups were not significant (Table 3). miR-210 expression in the severe clinical symptom group was significantly higher than that in the mild clinical symptom group.

Receiver operating characteristic (ROC) curve analysis of miR-210

The results of the ROC curve analysis showed that the area under the curve was 0.671 (0.526–0.816), which was statistically significant; thus, miR-210 appeared to possess a certain diagnostic value. The sensitivity of this diagnosis was 40.7%, and the specificity was 88.5% (Figure 3).

Figure 3 ROC curve analysis of miR-210. The diagnostic value of miR-210 was assessed by using a ROC curve. ROC, receiver operating characteristic.

The potential target genes of miR-210 were associated with neurodevelopmental and cardiovascular diseases

We also performed a bioinformatics analysis of the target genes of miR-210 involved in neurodevelopmental and cardiovascular diseases. A total of 278 miR-210 target genes were associated with cardiovascular diseases, 670 target genes were associated with neurodevelopmental diseases, and 142 target genes were associated with both neurodevelopmental and cardiovascular diseases (Figure 4A). In the BP domain, the top 10 GO pathways associated with neurodevelopmental diseases included nervous system development, neurogenesis, neuron part, and neuron generation, differentiation, and development, whereas the top three GO pathways associated with cardiovascular and cerebrovascular diseases included cardiovascular and circulatory system development and circulatory system processes (Figure 4B).

Figure 4 Analysis of the miR-210 potential target genes associated with neurodevelopmental and cardiovascular diseases. (A) Venn diagram depicting the overlap of the miR-210 target genes involved in neurodevelopmental and cardiovascular diseases. (B) GO analysis of the miR-210 potential target genes involved in the biological processes associated with neurodevelopmental and cardiovascular diseases. GO, Gene Ontology.

KEGG analysis of miR-210 target genes

We performed a KEGG pathway analysis of the target genes of miR-210 and found that the genes were mainly associated with metabolic pathways, cancer pathways, the phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (Akt) signaling pathway, endocytosis, and the mitogen-activated kinase-like protein (MAPK) signaling pathway (Figure 5A). The KEGG disease analysis revealed that the target genes of miR-210 were mainly associated with nervous system diseases, congenital metabolism disorders, musculoskeletal diseases, cancers, cardiovascular diseases, and immune system diseases (Figure 5B).

Figure 5 KEGG analysis of miR-210 target genes. (A) KEGG pathway analysis of the miR-210 target genes. (B) KEGG disease analysis of the miR-210 target genes. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Analysis of the target genes of miR-210 associated with the regulation of neurological or cardiovascular diseases

Further analyses showed that 102 miR-210 target genes were associated with autism (Figure 6A), and 26 miR-210 target genes were associated with epilepsy (Figure 6B). We analyzed the network interactions of the target genes of miR-210 involved in neurological or cardiovascular diseases. We then identified several potential target genes associated with miR-210 and neurological or cardiovascular diseases using the network diagram (Figure 7).

Figure 6 Analysis of the miR-210 target genes associated with autism and epilepsy. Venn diagram illustrating the miR-210 target genes in the GO category of nervous system development associated with autism (A) and epilepsy (B). GO, Gene Ontology.

Figure 7 Network interactions of miR-210 with the target genes involved in neurological or cardiovascular disease regulation.

Discussion

Neonatal asphyxia is a systematic syndrome of multiple organ damage due to hypoxemia, hypercapnia, and acidosis caused by an inability to breathe spontaneously after birth, and its pathogenesis may involve antepartum, intrapartum, or postpartum intrauterine hypoxia and respiratory and circulatory disturbances during childbirth (11). The incidence rate of neonatal asphyxia in China is 13.9%, and 15.6% of these patients suffer from disabilities of different degrees (12). Furthermore, 25–50% of disabled children with cerebral malaria aged under one year have HIE (13). Moreover, neonatal asphyxia and associated complications account for 33% of all neonatal deaths (14). Pathologically, hypoxia mainly leads to cerebral edema and neuronal necrosis, whereas ischemia prominently causes cerebrovascular infarction and white-matter softening (15). A retrospective study showed that the incidence of organ damage after asphyxia was 85.0%, whereas that of multiple organ damage was 57.5% (16). Brain injury is the main form of injury after asphyxiation.

No objective criteria exist for the diagnosis, grading, and prognosis evaluation of brain injury after neonatal asphyxia. Existing criteria include an abnormal obstetric history, umbilical artery blood gas analysis, and the Apgar scoring system. The sensitivity, specificity, and stability of evaluation criteria based on neurological symptoms and their duration, MRI findings, and NBNA scores after birth are unsatisfactory and associated with a significant time lag in assessing the occurrence and development of brain damage.

miRNA is a 22-nt long endogenous non-coding RNA that affects target mRNA translation via the complementary pairing of base sequences (7,17). Recently, numerous animal experiments have shown the involvement of miRNAs in the preconditioning regulation of cerebral hypoxia, protection of nerve cells, mechanisms related to ischemic nerve injury and apoptosis, and regulation of nerve regeneration after ischemia (18,19). The regulatory effects of miRNAs are characterized by their high evolutionary sequence conservation and strict spatiotemporal and tissue specificity, thus exerting effects before the occurrence of pathophysiological changes (20). Studies have shown the involvement of miRNAs in protecting nerve cells from ischemic preconditioning, regulating ischemic nerve injury- and apoptosis-related mechanisms, and regulating nerve regeneration after ischemia (21-23).

Recent studies have shown angiogenesis in the ischemic injury area after cerebral ischemia, and miR-210 is an ischemia-specific miRNA (10,24). Its expression is stably upregulated in the hypoxic environment in vitro (25). The miRNA can promote nerve function recovery in ischemic regions; however, its underlying regulatory mechanisms have not yet been elucidated. A study has shown that hypoxia upregulates miR-210 expression in human umbilical vein endothelial 12 (HUVE-12) cells, thereby promoting HUVE-12 cells to form blood vessels in vitro (26). We are the first to confirm the role of miR-210 in promoting angiogenesis under hypoxia in vitro, which is the main pathophysiological factor occurring after cerebral ischemia. Further research is necessary to determine if miR-210 participates in the regulation of neovascularization after the occurrence of cerebral ischemia (27).

Herein, we revealed that miR-210 was highly expressed in the peripheral blood of neonates with asphyxia and possessed certain diagnostic values for diagnosing neonatal asphyxia. Further, we found that high miR-210 expression in the peripheral blood was associated with a significant increase in the incidence of coma, hypotonia, and respiratory distress, indicating its involvement in the occurrence of brain hypoxia injury after asphyxia and was positively correlated with the clinical manifestations of brain injury. No statistically significant differences were observed in mild asphyxia symptoms with low specificity, such as clinical agitation and high muscle tension, between the two groups. Similarly, no statistically significant differences were found in abnormal WBC numbers, PCT levels, pH, NBNA scores, and abnormal imaging examinations between the two groups. These findings suggest that the protective mechanism of miR-210 is unrelated to the WBC and PCT systems.

A change in the pH of the umbilical cord blood indicates the blood pH of the body during a relatively short period at the time of delivery. Children with ischemia and hypoxia in the perinatal period might be treated effectively to reverse the internal environment of asphyxia and hypoxia before brain cell asphyxia damage occurs. Treatment conditions and individual differences in newborns’ tolerance to hypoxia affect the final degree of brain cell damage. The NBNA score partly reflects the recovery state of children after brain injury. Abnormal NBNA scores in the miR-210 high-expression group were lower than those in the miR-210 low-expression group. Despite the low P value, the results suggested that miR-210 expression was probably involved in brain protection. Nonetheless, future comprehensive studies with larger samples and multiple phases, such as 2 days and 7 days after asphyxia, need to be performed to verify these findings. For the in-depth analysis, the asphyxia group was divided into the normal and abnormal NBNA groups, normal and abnormal pH-value groups, and normal image examination and abnormal impact examination groups according to each clinical index for comparison; however, no significant differences were found in miR-210 expression among these groups. miR-210 expression in the severe clinical symptom group was notably higher than that in the mild clinical symptom group, and the difference was statistically significant, supporting the above results.

The bioinformatics analysis revealed that 142 target genes of miR-210 were associated with both neurodevelopmental and cardiovascular diseases, suggesting that miR-210 might participate in neurodevelopmental and cardiovascular disease progression. The miR-210 target genes were also associated with the metabolic, cancer, PI3K/Akt, and MAPK pathways, which can be examined as potential indicators while studying miR-210 target genes. Moreover, we also identified 102 and 26 miR-210 target genes associated with autism and epilepsy, respectively. The network interaction analysis showed miR-210 target genes associated with neurological or cardiovascular diseases. The autism- and epilepsy-related miR-210 target genes predicted based on the database require long-term follow-up and further investigation. Moreover, we plan to analyze more relevant clinical variables in our future study.

Conclusions

Based on the relationship between miR-210 expression and each clinical observation and prognosis, we suggest that miR-210 can be used as a new indicator of post-asphyxial hypoxic-ischemic brain injury. We found that these miR-210 target genes were associated with neurodevelopmental and cardiovascular diseases, as well as with autism and epilepsy. We further identified several miR-210 target genes associated with neurological or cardiovascular diseases using the PPI network maps. Thus, this study provides a basis for further research on using miR-210 in treating newborns with asphyxia and those with neurological or cardiovascular diseases.

Acknowledgments

Funding: This work was supported by the Dongguan Social Science and Technology Development Project (No. 2014108101011).

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-23-106/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-23-106/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-23-106/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-23-106/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted according to the Declaration of Helsinki (as revised in 2013). All the study participants’ parents signed an informed consent form consenting to their child’s inclusion in the study. This study was approved by the Ethics Committee of The Third People’s Hospital of Dongguan (No. 2022-KYSB-014).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)