Research subjects and clinical data collection

The present investigation received ethical clearance from the Research Ethics Board at The First Affiliated Hospital of Shihezi University, Xinjiang, China, all children’s legal guardians were informed and voluntarily signed the informed consent form. The study encompassed a sample of 169 preterm infants delivered at The First Affiliated Hospital of Shihezi University from 2019 to 2023, including 106 cases diagnosed with NRDS and an additional 63 healthy control subjects. The average gestational age for the control group and the NRDS group were 31.97 ± 2.28 weeks and 31.77 ± 2.25 weeks, respectively. The diagnosis of NRDS was primarily based on clinical manifestations and imaging results, characterized by a respiratory rate exceeding 60 breaths per minute, labored breathing, respiratory distress, cyanosis, and chest retractions in the intercostal or subcostal areas, or above the sternum. Additionally, imaging revealed bronchial obstruction, reticulated patterns, or areas of opacity in the lungs on X-ray examination. Patients harboring primary insufficiencies of alveolar surfactant, genetic congenital heart defects, preexisting metabolic irregularities, and anomalies of the pulmonary and thoracic walls were not included in the study. To exclude the influence of genetic conditions, we also referred to previous studies [17,18,19,20,21,22,23].

Basic data were collected, including gender, birth weight, gestational age, and blood gas oxygenation index. The basic data of the control group and the case group are shown in Table 1. There is no difference in gender, delivery weight and gestational age of newborns between the two groups, and the blood gas oxygenation index of patients in the NRDS group is significantly lower than that in the control group.

Table 1 Basic information statistics of subjectsNeonatal umbilical artery blood gas analysis

After birth and before the newborn’s first cry, two hemostatic clamps were used to clamp the proximal umbilical cord of the fetus, about 15 cm from the fetus’s navel, to block the blood circulation between the newborn and the placenta. Umbilical artery blood was collected using a heparinized syringe connected to the scalp needle. The umbilical artery blood gas of the newborn was examined by blood gas analyzer. The indicators include pH (Pondus Hydrogenii), PaO2 (Arterial Oxygen Partial Pressure), PaCO2 (Arterial Carbon Dioxide Partial Pressure), HCO3− (bicarbonate), and BE (Bicarbonate Excess). The whole test procedure should be completed within 30 min to preserve the integrity of the active components present in the newborn’s umbilical artery blood, thereby ensuring the accuracy of the test results.

Pulmonary ultrasonography

Each side of the lung was divided into anterior, lateral and posterior regions by parasternal line, anterior axillary line, posterior axillary line and posterior median line. Then, each lung is divided into two upper and lower regions by the middle line of the two nipples, making a total of 12 regions. The intercostal spaces were examined from the second intercostal space from the top to the bottom and from the inside to the outside with a line-array probe, followed by a parallel examination of each of the 12 regions of the lungs bilaterally, and the sonograms of each region of each side of the lung were stored and recorded. The scoring criteria refer to the criteria proposed by Rouby [24] and Sartori [25] et al. LUS score in the 12 regions of double lungs were the sum of the 12 regions, and the score ranged from 0 to 48 points.

Quantitative real-time PCR (qRT-PCR)

Neonatal venous blood was taken and centrifuged at a low temperature, then serum was collected. Total RNA was recovered from the blood serum using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). The quality and quantity of the isolated RNA were assessed using a NanoDrop 2000 spectrophotometer (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. Upon achieving an optical density ratio of A260/280 approximately equal to 2.0, the RNA was chosen for additional analysis. The isolated RNA was converted into complementary DNA (cDNA) through reverse transcription with the PrimeScript RT Reagent Kit (TaKaRa; Japan). The SYBR Green I Master Mix kit (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) was employed for qRT-PCR to determine the comparative expression levels of miR-513a-3p. The sequences of the primers were as follows:

miR513a-3p forward: 5’-TAAATTTCACCTTTCTGAGAAGG-3’,

miR-513a-3p reverse: 5’-GCGAGCACAGAATTAATACGAC-3’.

GAPDH forward: 5’-GGGAAACTGTGGCGTGAT-3’.

GAPDH reverse: 5’-GAGTGGGTGTCGCTGTTGA-3’.

Records of prognostic outcomes in the experimental group

The therapeutic outcomes at the 28-day mark were meticulously documented and categorized into two distinct categories. Favorable resolution or recovery: subsequent to the intervention, the neonate exhibited stable vital parameters, a notable reduction or complete resolution of respiratory disturbances, and a nearly standard chest radiograph. Consequently, the neonate was able to be weaned off mechanical ventilation and supplemental oxygen. Abandonment or death: therapeutic efforts were deemed unsuccessful, leading to persisting instability in the neonate’s vital signs. The neonate remained dependent on mechanical ventilation, encountered grave complications, or had treatment withdrawal requested by relatives. Consequently, the neonate was discharged from the healthcare facility or vital signs ceased, ultimately resulting in a clinical determination of death, often due to economic constraints or poor prognosis.

Statistical analysis

SPSS 26.0 and GraphPad Prism 9 were used for data processing and statistical evaluation. Data were expressed as mean ± standard deviation (SD) and independent sample t test was used. The logistic regression analysis was used to analyze the risk factors of poor prognosis in NRDS patients. ROC curve was used to evaluate the diagnostic ability of miR-513a-3p, arterial blood gas parameters and LUS score on NRDS and the predictive effect of prognosis. Pearson analysis was used for correlation analysis, and a p-value less than 0.05 was considered statistically significant.