Correlation of serum miR-203a-3p with the severity of neonates with necrotizing enterocolitis and analysis of its molecular mechanism

Study population

A total of 105 neonates with NEC who were hospitalized and treated in the author’s institution were enrolled as the study group. Additionally, 88 preterm infants without gastrointestinal disorders were selected as the control group during the same period. The sample size was determined by power analysis using G*Power 3.1 software, based on an effect size of d=0.5, with α=0.05 and power=90 %, yielding a minimum requirement of 64 subjects per group. Inclusion criteria for the study were as follows: 1) hospitalization at the same facility during the same period; 2) Availability of complete clinical information; 3) diagnosis and staging of NEC were strictly defined by Bell’s criteria as follows: Stage I (Suspected): temperature instability, apnea/bradycardia; abdominal distension; gastric residuals; occult blood in stool (no radiographic signs); Stage II (Definite): Stage I symptoms+radiographic signs (pneumatosis intestinalis, portal venous gas); thrombocytopenia; metabolic acidosis; Stage III (Advanced): Stage II symptoms+hemodynamic instability; intestinal perforation; abdominal tenderness/erythema; 4) for the control group: without gastrointestinal signs and symptoms, feeding intolerance, fecal and bowel abnormalities, or inherited metabolic disorders. Exclusion criteria included: 1) combined with digestive tract abnormalities; 2) with genetic metabolic disorders, chromosomal abnormalities, or immune deficiencies.

Peripheral blood of all participants was collected and subjected to centrifugation at 3,000 rpm for a duration of 10 min. Subsequent to this process, serum samples were preserved for the next experiments.

The study obtained the approval of the Ethics Committee of the author’s institution. All subjects’ legal guardians of all subjects involved have given informed consent.

Cell cultivation and transfection

The human normal colorectal fetal human colon (FHC) cells, purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), were maintained in a controlled environment at 37 °C and 5 % CO₂. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % serum fetal bovines was used as the culture medium. 1 % penicillin-streptomycin was added to the medium. FHC cells were treated with lipopolysaccharide (LPS) at different concentrations (0, 5, 10, 20, and 50 μg/mL) for 3 and 6 h, and the optimal LPS concentration was screened to establish the NEC cell model.

Lipofectamine 3,000 (Invitrogen) was utilized for transfecting miR-203a-3p mimic/mimic-NC, miR-203a-3p inhibitor/inhibitor-NC into FHC cells. Subsequently, the cells were stimulated with LPS for 4 h.

qRT-PCR assay

Based on the instruction provided by the manufacturer, TRIzol reagent was utilized for the extraction of total RNA from serum and cells. cDNA was reservedly transcribed with a TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Shanghai, China). The SYBR Premix Ex Taq II was employed for the detection of qRT-PCR on the BIO-RAD 7500 System. RNU6B was taken for the normalization of miR-203a-3p. Relative abundances of miR-203a-3p were computed by means of the equation of 2^−ΔΔCT approaches. Three replications were carried out for each reaction.

Analysis of inflammatory mediators

Cell supernatants were obtained for the measurement of IL-6 (interleukin-6), IL-8, and IL-1β levels using the corresponding ELISA Kits (Beyotime, Beijing, China). The detection of the inflammatory mediators was carried out based on the direction of the manufacturer. The optical density (OD) values were calculated at 450 nm by a microplate reader.

Redox-stress response test

Furthermore, superoxide dismutase (SOD) and catalase (CAT) testing reagents (Biological Engineering Research Institute, Nanjing, China) were obtained for the detection of SOD and CAT levels in the cell supernatant following the manufacturers’ directions.

Flow cytometry assay

Flow cytometry (BD Biosciences, Heidelberg, Germany) was utilized for testing cell apoptosis. The cells were washed with PBS and re-suspended in phosphate-buffered saline (PBS). Annexin V-FITC (fluorescein isothiocyanate) and PI (propyl iodide) were added to the cell suspension and cultivated for 30 min at 4 °C in the dark. The apoptosis was monitored using FACScan flow cytometry (BD Biosciences).

Measurement of cell viability

MCF-7 cells were inoculated in 96-well plates and cultured overnight to allow for cell adhesion to the wall. At 48 h, 10 μL of CCK-8 reagent was added, and incubation continued for 1 h. After the incubation period, the cells were protected from light, and the absorbance at 450 nm was detected by an enzyme marker. Three wells were established for each group, and the cell proliferation viability of each group was quantified by the absorbance value.

Dual-luciferase reporter assay

The 3′-untranslated regions (3′-UTR) of ATM-WT and ATM-MUT of ATM were cloned and inserted into pmiRGLO vector and then co-transfected with miR-203a-3p mimic/inhibitor into FHC cells. After 48 h transfection, the cells were lysed and luciferase activity was detected with Dual luciferase Assay Kit (Vazyme, Nanjing, China). Luciferase activities were tested using a microplate reader, which was standardized by sea renilla luciferase.

Bioinformatic analysis

The miRPathDB, miRDB, and miRWalk databases were utilized for the prediction of miR-203a-3p target genes, and these miR-203a-3p-related genes were analyzed with Venn diagrams. Meanwhile, for the miR-203a-3p-regulated genes, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were executed for the exploration of potential molecular function and associated signaling pathways. As for the protein analysis, the Search Tool of the Retrieval of Interacting Genes/Proteins (STRING) database was employed for the online protein-protein interaction (PPI) prediction analysis.

Statistics

The data analysis was performed with the utilization of the statistical software package SPSS 23.0 in conjunction with the graphing software GraphPad Prism 7.0. The continuous variables were averaged and the standard deviation (SD) was computed accordingly. Student’s t-test was executed to examine two-group disparities, while a one-way ANOVA test was conducted for comparisons involving three or more groups. The association of categorical variables between the two groups was assessed using the Chi-square test. Logistic regression analysis was applied to identify risk factors for NEC. Additionally, receiver operating characteristic (ROC) curves were generated to ascertain the diagnostic property of miR-203a-3p. The relevance of miR-203a-3p with ATM was assessed using Pearson’s correlation coefficient analysis. A p value of less than 0.05 indicated the presence of a statistically significant difference.

Baseline data of neonates with NEC and controls

As illustrated in Table 1, the fundamental clinical characteristics, including sex, gestational age, weight, delivery mode, feeding patterns, as well as laboratory parameters such as white blood cells (WBC), neutrophil (NEU), platelets (PLT), c-reactive protein (CRP), procalcitonin (PCT), albumin (ALB), were compared between neonates with NEC group and control group. Inter-group comparisons revealed no obvious differences in sex, gestational age, and weight of the study group and controls (all p>0.05). In contrast, the NEC group exhibited a notable decrease in the levels of WBC, NEU, PLT, and ALB when compared to the control group, suggesting a statistical significance (all p<0.05). Conversely, in the neonates with NEC, CRP and PCT levels were distinctly enhanced relative to their non-NEC counterparts (all p<0.001). Furthermore, a notable association was identified regarding delivery mode and feeding patterns between the two groups (all p<0.05). Additionally, it was observed that 60 (57.1 %) neonates with NEC were classified at Bell stage I, while 45 (42.9 %) neonates were categorized at Bell stage II/III.

Relative abundances of miR-203a-3p in neonates with NEC

To investigate the expression pattern and diagnostic potential of miR-203a-3p in neonates suffering from NEC, qRT-PCR assay was executed. The results demonstrated a significant reduction in miR-203a-3p levels in the NEC cohort against controls (p<0.001, Figure 1A). Furthermore, the AUC was determined to be 0.928, effectively differentiating between neonates with NEC and those with non-NEC, exhibiting a sensitivity of 80.7 % and a specificity of 92.4 % (Figure 1B).

Figure 1:

Relative abundances of miR-203a-3p in neonates with NEC. A. Serum miR-203a-3p was distinctly declined in neonates with NEC relative to control subjects (p<0.001). B. The AUC of miR-203a-3p was 0.928, highlighting its robust efficacy in discriminating neonates with NEC from those with non-NEC. C. A pronounced decrease in serum miR-203a-3p was found in neonates at the bell stage I and II/III compared to controls (p<0.001). D. A substantial diagnostic capability of serum miR-203a-3p was found in differentiating neonates at the bell stage II/III from those at the bell stage I. The AUC was 0.803 (NEC: Necrotizing enterocolitis; AUC: Area under the curve; ***: p<0.001 NEC vs. controls; ###: p<0.001 bell stage II/III vs. Bell stage I).

For the subgroup analysis, the NEC group was stratified into Bell stage I and Bell stage II/III subgroups. When compared to the control group, miR-203a-3p expression exhibited a significant decline across both the Bell stage I and Bell stage II/III groups, respectively (p<0.001). Notably, a substantial reduction in miR-203a-3p levels was noted in neonates classified within the Bell stage II/III subgroup, in contrast to those in Bell stage I (p<0.001, Figure 1C). Additionally, the diagnostic capacity of miR-203a-3p in differentiating neonates in the Bell stage II/III and those in the Bell stage I was evaluated using ROC analysis. The findings revealed a robust diagnostic potential, reflected by an AUC of 0.803. The sensitivity was 75.0 % and the specificity was 75.6 % (Figure 1D).

Comparison analysis of clinical information of different bell stages in neonates with NEC

Based on the Bell classification criteria, a cohort of 105 neonates with NEC was split into two groups: Bell stage I (n=60) and stage II/III (n=45). As detailed in Table 2, a statistically significant association was found across several clinical parameters, including gestational age (p<0.001), WBC (p=0.016), CRP (p=0.003), PCT (p<0.001) and ALB (p=0.009) when comparing the Bell stage I to stages II/III. Nevertheless, no statistically significant differences were found regarding sex, weight, delivery mode, feeding patterns, NEU, and PLT between the Bell stage I and stages II/III of neonates with NEC, as all comparisons yielding p>0.05.

Risk factors analysis for NEC bell stages

To evaluate the influencing factors for the severity of NEC as classified by Bell stages, logistic regression analysis was conducted utilizing the clinical information. An examination of the univariate regression results for Bell stage I and II/III in neonates with NEC revealed that several significant associations pertaining to the progression towards more severe stages of the condition. Specifically, gestational age (p<0.001; OR=0.182; 95%CI=0.078–0.426), WBC (p=0.006; OR=0.323; 95%CI=0.143–0.728), CRP (p=0.037; OR=2.321; 95%CI=1.052–5.125), ALB (p=0.027; OR=0.275; 95%CI=0.088–0.863) and miR-203a-3p (p=0.001; OR=0.234; 95%CI=0.096–0.569) emerged as highly significant predictors (Table 3).

The factors associated with the condition were incorporated into a multivariate logistic regression analysis. The results showed that miR-203a-3p (p=0.006; OR=0.168; 95%CI=0.055–0.514) emerged as a significant independent risk indicator for NEC Bell stage II/III. Additionally, gestational age (p=0.001; OR=0.203; 95%CI=0.076–0.540), WBC (p=0.021; OR=0.303; 95%CI=0.109–0.838) and ALB (p=0.046; OR=0.341; 95%CI=0.119–0.979) were also identified as critical contributors to the risk assessment (Table 3).

Influences of miR-203a-3p on cellular activities

To establish the NEC cell model, five distinct concentrations of LPS, namely 0, 5, 10, 20, and 50 μg per milliliter, were utilized to stimulate FHC cells. After qRT-PCR examination, it was observed that the levels of miR-203a-3p levels progressively decreased in FHC cells subjected to increasing LPS concentrations (0, 5, 10, 20, and 50 μg/mL) over 3 and 6 h in respective order (p<0.001, Figure 2A). Moreover, following the transfection with miR-203a-3p mimic, a substantial increase in miR-203a-3p levels was noted in comparison to the mimic-NC group, indicating a high transfection efficiency (p<0.001). Notably, upon LPS treatment, miR-203a-3p expression in FHC cells was distinctly suppressed; however, this decrease was effectively mitigated by transfection with the miR-203a-3p mimic (Figure 2B).

Figure 2:

Influences of miR-203a-3p on cellular functions in an LPS-induced cell model. A. MiR-203a-3p expression was quantified in FHC cells subjected to varying concentrations of LPS (0, 5, 10, 20, and 50 μg/mL). B. Following transfection with miR-203a-3p, a significant increase in miR-203a-3p levels was observed in FHC cells relative to controls, confirming the efficacy of the transfection. MiR-203a-3p was declined in LPS-triggered cells, a decline that was successfully reversed in the LPS+miR-203a-3p mimic group. C. The assessment of cell apoptosis was conducted in LPS-triggered cells post-transfection with miR-203a-3p. D. In the NEC cell model, cell viability was evaluated after transfection with miR-203a-3p mimic. E. Inflammatory indicators were detected and analyzed across different experimental groups. F. The redox-stress response, including SOD and CAT enzymatic activities, was determined in the NEC cell model after transfection with the miR-203a-3p mimic (NEC: Necrotizing enterocolitis; LPS: Lipopolysaccharide; FHC: Fetal human cells; SOD: Superoxide dismutase; CAT: Catalase; ***: p<0.001 against mimic-NC/inhibitor-NC/controls; ###: p<0.001 against LPS+mimic-NC).

Furthermore, the evaluation of cell apoptosis and viability in FHC cells were conducted following transfection with the miR-203a-3p mimic and stimulation with LPS. Notably, the rate of cell apoptosis showed a significant increase in the LPS group compared to the control group, a phenomenon that was markedly alleviated by transfection with the miR-203a-3p mimic (p<0.001, Figure 2C). Regarding cell viability, a pronounced inhibition of cell growth was observed in the LPS group in contrast to the control group (p<0.001), with this detrimental effect similarly alleviated post-transfection with the miR-203a-3p mimic (Figure 2D). Concurrently, the levels of pertinent inflammatory indicators and antioxidant enzyme activities were assessed within the NEC cell model. The LPS-stimulated cells exhibited a substantial increase in levels of IL-6, IL-8, and IL-1β relative to control cells (p<0.001), with this elevation significantly diminishing following miR-203a-3p transfection (p<0.001, Figure 2E). As illustrated in Figure 2F (p<0.001), a significant decrease in the activities of SOD and CAT was detected in the LPS group relative to controls (p<0.001). In contrast to the LPS+mimic-NC group, the enzyme liveness of SOD and CAT was conspicuously augmented in the LPS+miR-203a-3p mimic group (p<0.001). This finding suggested that the reduction in SOD and CAT activities within the NEC cell model could be effectively counteracted by transfection with the miR-203a-3p mimic.

Bioinformatic analysis of miR-203a-3p-associated genes

Based on the prediction analysis conducted using the miRPathDB (8,765 predicted genes), miRDB (1,352 predicted genes), and miRWalk (1,393 predicted genes) databases, a Venn diagram was employed to ascertain the intersection of the three databases, revealing a total of 144 overlapping genes (Figure 3A). Moreover, GO and KEGG analyses were employed for the exploration of the functional roles and pathways associated with the predicted genes of miR-203a-3p. GO analysis demonstrated that the functions of miR-203a-3p-related genes primarily encompassed several critical domains: regulation of GTPase activity, histone mRNA catabolic process, and regulation of signaling receptor activity of biological process (BP); cation channel complex, guanyl-nucleotide exchange factor complex, ion channel complex of cellular component (CC); diacylglycerol kinase activity, ras guanyl-nucleotide exchange factor activity, NAD₊ kinase activity of molecular function (MF) (Figure 3B). Concurrently, KEGG pathway analysis was conducted to gain insights into gene function, highlighting that the most significantly enriched pathways included the cAMP signaling pathway, choline metabolism in cancer, C-type lectin receptor signaling pathway, and so on (Figure 3C). Notably, of the aforementioned pathways, the MAPK signaling pathway exhibited the strongest correlation with NEC. These mined BP, CC, and MF terms and pathways are intricately associated with the pathophysiology of NEC.

Figure 3:

Bioinformatic analysis of miR-203a-3p-regulated genes. A. A venn diagram illustrated the overlapping genes affected by miR-203a-3p, as identified through the miRPathDB, miRDB, and miRWalk repositories. B. Gene ontology (GO) analysis of miR-203a-3p-regulated genes. C. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of miR-203a-3p-regulated genes.

Verification of target interaction of miR-203a-3p with ATM

To gain a deeper understanding of the potential molecular mechanisms through which miR-203a-3p influenced the pathogenesis of NEC, ATM was selected for further investigation in subsequent experiments, guided by bioinformatic analysis. The target sequences of miR-203a-3p with ATM were predicted via miRPathDB, miRDB, and miRWalk algorithms, as depicted in Figure 4A. In comparison to the mimic-NC group, a significant reduction in luciferase activities was observed in the WT-ATM group upon transfection with the miR-203a-3p mimic, indicating a remarkable enhancement in luciferase liveness (p<0.001). Conversely, no significant alterations of luciferase activities were noted in the MUT-ATM group (p<0.001, Figure 4B). Moreover, analysis of serum samples from neonates with NEC revealed a marked elevation of ATM levels when compared to control groups (p<0.001, Figure 4C). The relevance between serum miR-203a-3p and ATM was subsequently examined, revealing a negative correlation (r=−0.688, p<0.001, Figure 4D). Additionally, the relative expression of ATM across various experimental groups, including blank control, LPS, LPS+mimic-NC, and LPS+miR-203a-3p mimic groups, was investigated. Results indicated a substantial increase in ATM levels in the LPS group against the blank control group (p<0.001). Notably, an obvious reduction of ATM level was discovered in FHC cells following transfection with miR-203a-3p and subsequent LPS stimulation (p<0.001, Figure 4E).

Figure 4:

Illustration of ATM targeting by miR-203a-3p. A. The binding sites of miR-203a-3p and ATM were presented. B. Results from a luciferase assay conducted on FHC cells co-transfected with either ATM-WT or ATM-MUT reporter plasmid in conjunction with miR-203a-3p mimic or inhibitor. C. Measurement of the relative abundances of ATM in the serum of neonates with NEC and controls. D. Identification of a statistically significant negative correlation between miR-203a-3p and ATM, evidenced by a correlation coefficient of r=−0.688 (p<0.001). E. In LPS-treated cells, a notable increase in ATM expression was observed relative to controls, a phenomenon that was effectively mitigated upon transfection with miR-203a-3p mimic (NEC: Necrotizing enterocolitis; ATM: Ataxia telangiectasia mutated; ***: p<0.001 against mimic-NC/inhibitor-NC/controls; ###: p<0.001 against LPS+mimic-NC).