Identification of serum miR‑320a in gestational diabetes mellitus for predicting macrosomia and in vitro mechanistic investigation

Abstract

Background

Gestational diabetes mellitus (GDM) significantly elevates the risk for a spectrum of adverse pregnancy outcomes. The expression pattern and regulatory mechanism of microRNA-320a (miR-320a) in GDM remain poorly elucidated.

Methods

The study enrolled 40 GDM patients along with 40 healthy controls. Serum expression levels of miR-320a were quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR). ROC curve analysis and binary logistic regression were applied to evaluate its diagnostic performance for GDM and predictive value for fetal macrosomia, respectively. In HTR-8/SVneo trophoblast cells, CCK-8 assay and flow cytometry were used to assess the influence of miR-320a on cell proliferation and apoptosis. Dual-luciferase reporter assay validated the direct targeting relationship between miR-320a and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD). Western blot was performed to detect PIK3CD protein expression, and rescue experiments further explored the biological function of the miR-320a/PIK3CD axis in trophoblast proliferation and apoptosis.

Results

Serum miR-320a was significantly downregulated in GDM patients and exhibited good diagnostic capability to distinguish GDM from healthy pregnancies. Lower miR-320a expression was correlated with a higher risk of fetal macrosomia. In addition, overexpression of miR-320a promoted cell proliferation and suppressed apoptosis in HTR‑8/SVneo cells. PIK3CD was identified as a direct downstream target of miR‑320a, and miR‑320a overexpression inhibited PIK3CD expression. Furthermore, the effects of miR‑320a on cell proliferation and apoptosis were markedly reversed by co‑transfection with pcDNA‑PIK3CD.

Conclusions

Circulating miR-320a has promising potential for GDM diagnosis and fetal macrosomia prediction. Increased miR-320a expression facilitates proliferation and restricts apoptosis in HTR-8/SVneo trophoblast cells by directly targeting PIK3CD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12920-026-02401-x.

Background

Diabetes mellitus ranks among the most prevalent metabolic disorders worldwide, with the affected population projected to exceed 400 million globally [1]. Gestational diabetes mellitus (GDM) is a metabolic disorder of pregnancy driven by an imbalance between the degree of insulin resistance and the β-cell secretory capacity, culminating in pathological hyperglycemia, and poses a significant health concern for pregnant women, affecting 4%-12% of pregnancies [2]. GDM is closely linked to an elevated risk of adverse pregnancy outcomes, with multifaceted negative impacts on maternal and fetal health, including preterm birth, hypertension, delivery-related trauma, cesarean section, congenital anomalies, macrosomia, and intrauterine growth restriction [3]. Neonates born to mothers with GDM are at an elevated risk for complications associated with fetal hyperinsulinemia, including neonatal hypoglycemia, respiratory disorders such as transient tachypnea of the newborn (TTN), and hypocalcemia [4]. Severe neonatal hypoglycemia can even increase the risk of permanent brain damage [5]. Macrosomia also raises the likelihood of delivery-related injuries and difficult labor, potentially resulting in neonatal asphyxia [6]. Given these risks, it is important to find biomarkers that can effectively monitor GDM to predict adverse pregnancy outcomes.

MicroRNAs (miRNAs) are evolutionarily conserved short non-coding RNAs (17–22 nucleotides) [7]. These molecules regulate gene expression through sequence-specific binding to the 3’-untranslated regions (3’-UTRs) of target mRNAs, triggering translational repression or mRNA degradation [8], and are important epigenetic factors. A single miRNA can regulate dozens or even hundreds of mRNAs [9, 10], consequently exerting pivotal regulatory influences across diverse biological systems and disease pathologies. Moreover, miRNAs can be detected in a wide range of body fluids, which makes them attractive potential biomarkers [11]. In diabetes, several miRNAs have been identified to play key roles. For example, through targeted suppression of KLF2, miR-92a preserves pancreatic β-cell functionality in diabetic individuals, whereas overexpression of miR-708 induces β-cell apoptosis [12, 13]. In addition, studies have revealed that miR-320a can exacerbate diabetic nephropathy by inhibiting MafB and may serve as a potential therapeutic target for this disease [14].

Phosphatidylinositol 3‑kinases (PI3Ks) constitute a family of lipid kinases involved in the modulation of a wide range of cellular processes, including cell growth, proliferation, differentiation, and intracellular trafficking [15]. Class IA PI3Ks function as heterodimers consisting of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. To date, three distinct isoforms of the catalytic subunit have been identified, namely PI3Kα, PI3Kβ, and PI3Kδ, which are encoded by the PIK3CA, PIK3CB, and PIK3CD genes, respectively. Among these, PI3Kδ is predominantly expressed in leukocytes and serves as a critical mediator in both innate and adaptive immune responses [16]. Additionally, elevated PIK3CD mRNA expression has been detected in women diagnosed with GDM diagnosed later in pregnancy, and this gene is significantly enriched in several diabetes‑related signaling pathways, suggesting that it may contribute to the pathological processes underlying type 2 diabetes [17].

This study investigated the role of miR‑320a in GDM and demonstrated that this molecule may attenuate GDM progression by targeting PIK3CD. Together, these results highlight a promising molecular target for the diagnosis and intervention of GDM.

Methods

Bioinformatics analysis

The miRDB database (https://mirdb.org/) was used to predict the target genes of miR-320a, and the GeneCards database (https://www.genecards.org/), a comprehensive database for collecting and annotating human disease-associated genes, was employed to screen genes associated with GDM. Venn diagram analysis was then performed to identify the intersection of miR‑320a target genes and GDM‑related genes, from which PIK3CD was selected for further investigation.

Sample size calculation

A priori sample size calculation was performed using G*Power 3.1 software. An effect size of 0.55, type I error rate (α) of 0.05, and power (1 − β) of 0.95 were set for a two-group comparison. The calculated minimum sample size was 33 participants per group. To ensure statistical robustness, 40 GDM patients and 40 healthy controls were ultimately included in this study.

Patient recruitment and sample collection

We recruited pregnant women aged 25–45 without diabetes for prenatal examination. Those with pre-pregnancy diabetes or conditions such as hypertension, nephropathy, hepatitis, bacterial/viral infections, or inflammation were excluded. Initially, 40 patients with GDM were enrolled. 40 healthy controls matched for both age and gestational age were concurrently recruited. Written informed consent was obtained from all participants. GDM diagnosis was established by oral glucose tolerance test (OGTT) at 24–26 weeks of gestation. Fasting peripheral blood samples were collected from participants in both groups. The upper layer of serum was separated, carefully transferred to new sterile EP tubes, labeled, and stored in a -80 °C freezer. All serum samples were visually inspected for hemolysis prior to RNA extraction, and samples with obvious hemolysis were excluded to avoid confounding effects on miRNA quantification. Additionally, data on birth weight were gathered for the neonates. A birth weight greater than the 90th percentile for gestational age, according to local population growth curve standards, is defined as fetal macrosomia.

Cell culture

HTR-8/SVneo cells (obtained from the Cell Bank of the Chinese Academy of Sciences) were maintained at 37 °C in a humidified 5% CO₂ atmosphere. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 25 mmol/L glucose (Sigma-Aldrich).

MiRNA mimics, miRNA inhibitors, pcDNA-NC, pcDNA-PIK3CD, and transfection experiments

All expression plasmids—including the non-targeting negative control (pcDNA-NC) and PIK3CD-targeting construct (pcDNA-PIK3CD)—were commercially obtained from GenePharma (China). Similarly, miR-320a mimic and inhibitor reagents, alongside their corresponding non-targeting negative controls, were sourced from the same supplier. HTR-8/SVneo cells were seeded in culture plates and grown to 70–80% confluence before transfection. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The final concentration of miR-320a mimic, inhibitor, and negative controls was 50 nM. For plasmid transfection, 2 µg of pcDNA-NC or pcDNA-PIK3CD was used per well. After 6 h of incubation, the medium was replaced with fresh complete medium, and cells were cultured for another 48 h before subsequent experiments.

Cell counting kit-8 (CCK-8) assay

Cellular viability was assessed using the CCK8 assay kit (Beyotime Biotechnology, C0038). Cells were seeded in a 96-well plate at 100 µL per well and subjected to various treatments as per the experimental design. Absorbance measurements at 450 nm, performed on a Thermo Fisher Scientific microplate reader, were used to quantify cell viability.

Cell apoptosis by flow cytometry

Cells were resuspended in binding buffer containing FITC-conjugated Annexin V and propidium iodide (PI) for apoptosis detection. Flow cytometry (BD Biosciences) was used to analyze apoptotic cells.

Dual luciferase reporter gene experiment

The reporter plasmids, including wild-type (PIK3CD-WT) and mutant (PIK3CD-MUT), were generated by inserting the PIK3CD 3’-UTR fragment into the pmirGLO dual-luciferase reporter vector (Invitrogen, USA). Subsequently, HTR-8/SVneo cells were prepared and co-transfected using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015). Transfection mixtures included either the PIK3CD-WT or PIK3CD-MUT reporter plasmid, in combination with either mimics-miR (experimental) or its negative control mimics-NC, alongside either specific inhibitors (experimental) or their negative control (inhibitor-NC). After 48 h of incubation, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, E1910). Measurements were performed sequentially, first detecting Firefly luciferase luminescence followed by Renilla luciferase luminescence.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

For qPCR analysis, cDNA (1 µL) synthesized from total RNA (isolated using the Beyotime RNA/Protein Isolation Kit, R0018S per manufacturer’s instructions) was amplified with Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, 4367659) on a QuantStudio Dx Real-Time PCR System. Reverse transcription employed the PrimeScript™ RT Reagent Kit (Takara, RR037Q). The relative expression of target genes was determined using the 2^–ΔΔCt method, and each sample was tested in triplicate to reduce experimental error. β-Actin and U6 were used as the endogenous reference genes for mRNA and miRNA quantification, respectively. For serum miRNA quantification, U6 snRNA was initially selected as the internal reference based on its widespread use in previous GDM-related circulating miRNA studies [18, 19]. To formally verify the expression stability of U6 in all enrolled samples, we evaluated its stability by calculating the standard deviation (SD) of raw Ct values. According to widely recognized criteria, Ct SD values ranging from 0.5 to 1.0 represent moderate expression stability. The results showed that the Ct value SD of U6 fell within this range, indicating its mild and acceptable expression fluctuation among all samples. Collectively, U6 was confirmed to be stably expressed and suitable to serve as a reliable internal reference gene for serum miRNA relative expression quantification in this study. The specific primer sequences were as follows: PIK3CD, forward 5’-CCCACATGAAGAGGAACTGAGAT-3’, reverse 5’-AGTCACTGAGCCTGCCAACC-3’; β-Actin, forward 5’-GCCAACACAGTGCTGTCTGG-3’, reverse 5’-GCAGAGGAGGCAATGATCTTG-3’; U6, forward 5’-GCTTCGGCAGCACATATACTAAAAT-3’, reverse 5’-CGCTTCACGAATTTGCGTGTCAT-3’; miR-320a, forward 5’-TCGGCAGGGCCTTCTCTTCCCG-3’, reverse 5’-CAGTGCAGGGTCCGAGGT-3’.

Western blot analysis

Total cellular protein was extracted from each group, and protein concentrations were determined using a BCA Protein Quantification Kit (Beyotime, P0010) to ensure equal loading. Equal amounts of protein samples were mixed with 5× SDS-PAGE loading buffer, denatured at 95 °C for 5 min, and separated by 10% SDS-PAGE (80 V for the stacking gel, 120 V for the separating gel, approximately 90 min). Proteins were then transferred onto a PVDF membrane (Millipore, IPVH00010) using the wet transfer method at a constant current of 300 mA at 4 °C for 120 min. The membrane was blocked with 5% non-fat milk (Beyotime, P0216) for 1 h at room temperature to reduce non-specific binding. After washing three times with TBST buffer (10 min each), the membrane was incubated with the primary antibody against PIK3CD (Proteintech, 67964-1-Ig; PI3 Kinase p110 Delta Monoclonal antibody), diluted 1:5000 in primary antibody diluent (Beyotime, P0256), for 1.5 h at room temperature. Following another three washes with TBST, the membrane was incubated with the corresponding horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Proteintech, PR30012), diluted 1:10000 in secondary antibody diluent (Beyotime, P0258), for 1 h at room temperature. After three final washes with TBST, immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher, 34577). Band intensities were quantified using ImageJ software, and the relative expression level of PIK3CD was calculated as the ratio of the target protein intensity to that of the internal reference β-actin. All experiments were independently repeated three times.

Statistical analysis

Data were presented as the mean ± SD from at least three independent experiments. Categorical variables were analyzed using the chi-square test. Normality of the data distribution was assessed using the Shapiro-Wilk test. Inter-group comparisons were performed using Student’s t-test for normally distributed continuous variables and the Mann-Whitney U test for non-normally distributed continuous variables. Statistical analyses were conducted using GraphPad Prism 9.3.1 and IBM SPSS Statistics 23. Receiver operating characteristic (ROC) curve analysis was performed using GraphPad Prism 9.3.1, and the area under the curve (AUC) with 95% confidence interval (95% CI), sensitivity, and specificity were calculated. Binary logistic regression was performed using IBM SPSS Statistics 23 to evaluate the association between miR‑320a and fetal macrosomia; the Hosmer‑Lemeshow test was used for model fitness, and odds ratios (OR) with 95% CI were presented. A two‑sided p < 0.05 was considered statistically significant.

Results

MiR-320a has potential diagnostic value in GDM

This study involved 40 GDM patients and 40 age-and gestational age-matched healthy controls. Fasting peripheral blood samples were collected to measure serum miR-320a levels. The stability of the reference gene U6 used for miRNA quantification in these samples was validated by calculating the standard deviation of raw Ct values. Results showed that compared to the control group (NGT group), miR-320a expression was significantly lower in the GDM group (Fig. 1A). The diagnostic utility of miR-320a for GDM was subsequently evaluated using ROC curve analysis, which showed an AUC of 0.9406 (p < 0.001, Fig. 1B), with a sensitivity of 87.50% and a specificity of 87.50%, indicating that miR-320a can effectively distinguish between healthy and GDM pregnant women. Patients were divided into low and high miR‑320a expression groups according to the mean expression value of miR‑320a in the GDM patient group. Samples with expression levels higher than the mean were assigned to the high miR‑320a expression group, and those lower than the mean were assigned to the low expression group. The association between miR‑320a expression and various clinicopathological parameters, as well as neonatal birth weight, was subsequently analyzed (Table 1). The results showed that miR‑320a expression levels were significantly correlated with neonatal birth weight, fasting blood glucose (FBG), 2-hour OGTT plasma glucose values, and hemoglobin A1c (HbA1c). Binary logistic regression analysis revealed FBG was a risk factor for fetal macrosomia. Furthermore, there was a significant association between serum miR‑320a levels and the risk of fetal macrosomia (Table 2). Specifically, low miR‑320a expression was associated with an increased risk of fetal macrosomia, with an OR of 0.148 and a 95% CI of 0.027–0.816 (p = 0.028). The Hosmer‑Lemeshow test confirmed good model fitness (p = 0.389), indicating that the regression model adequately fit the data. This suggests miR-320a may influence this critical pregnancy outcome and serve as a promising diagnostic biomarker for GDM as well as a valuable predictor for neonatal birth weight.

Fig. 1.

miR-320a expression in gestational diabetes mellitus and ROC curve analysis. A Assessment of miR-320a expression in serum samples from healthy pregnant women versus pregnant women diagnosed with gestational diabetes mellitus. B ROC curve analysis of miR-320a. ***p < 0.001

Table 1.

The correlation between serum miR-320a and clinical characteristics

Characteristics	Case (n = 40)	miR-320a		p value
		low	high
Age				0.152
< 35 (years)	22	12	10
≥ 35 (years)	18	11	7
Triglycerides in the early stage of pregnancy				0.980
< 1.2 (mmol/L)	17	10	7
≥ 1.2 (mmol/L)	23	13	10
Pre-pregnancy BMI				0.148
< 21.2 (kg/m2)	17	9	8
≥ 21.2 (kg/m2)	23	14	9
Gestational weight gain				0.750
< 11.2 (kg)	21	13	8
≥ 11.2 (kg)	19	10	9
Gestational age				0.342
≥ 39.5(week)	19	10	9
< 39.5(week)	21	13	8
Neonatal birth weight				0.005**
< 4 (kg)	14	7	7
≥ 4 (kg)	26	16	10
Fasting blood glucose				0.010*
< 5.2 (mmol/L)	18	6	12
≥ 5.2 (mmol/L)	22	17	5
2-hour OGTT values				0.012*
< 7.5 (mmol/L)	29	13	16
≥ 7.5 (mmol/L)	11	10	1
HbA1c				0.007**
< 4.4 (%)	25	10	15
≥ 4.4 (%)	15	13	2

BMI body mass index, 2-hour OGTT values 2-hour oral glucose tolerance test plasma glucose values, HbA1c hemoglobin A1c

*p < 0.05; **p < 0.01

Table 2.

A binary logistics analysis was conducted to assess the significance of miR-320a in predicting fetal macrosomia

Parameters	OR	95% CI	p
Age (years)	1.045	0.168–6.483	0.962
Triglycerides in the early stage of pregnancy (mmol/L)	1.637	0.295-9.100	0.573
Pre-pregnancy BMI (kg/m2)	1.078	0.174–6.665	0.936
Gestational age (week)	2.474	0.367–16.662	0.352
Gestational weight gain (kg)	1.654	0.282–9.683	0.577
Fasting blood glucose (mmol/L)	6.089	1.012–36.648	0.049*
miR-320a	0.148	0.027–0.816	0.028*

OR odds ratio, 95% CI 95% confidence interval, BMI body mass index

*p < 0.05

Upregulation of miR-320a can inhibit the progression of HG-induced cellular injury

HTR-8/SVneo cells were used to simulate the pathological microenvironment of GDM via stimulation with 25 mmol/L high glucose (HG), with the aim of investigating the effect of miR-320a on cellular behaviors. Transfection results indicated that miR-320a expression was notably higher in the HG + miR-320a mimics group compared to the HG + mimics-NC and HG + inhibitor-NC groups, while it was markedly lower in the HG + miR-320a inhibitor group. These findings confirmed the successful overexpression and knockdown of miR-320a following transfection with miR-320a mimic and inhibitor, respectively (Fig. 2A).

Fig. 2.

Impact of miR-320a expression on HTR-8/SVneo cell proliferation and apoptosis. A miR-320a mRNA levels across experimental groups. B Cell proliferation assessed by CCK-8 assay following miR-320a modulation. C Apoptosis rate quantified by flow cytometry after miR-320a manipulation. **p < 0.01; ***p < 0.001

Subsequent functional experiments showed that, at all detected time points, cell viability was higher in the miR-320a mimics group than in the mimic-NC group, whereas the miR-320a inhibitor group exhibited lower cell viability relative to the inhibitor-NC group (Fig. 2B). These results suggest that overexpression of miR-320a may promote cell proliferation, while its knockdown may have an inhibitory effect on this process. In addition, apoptosis assays revealed that the apoptosis rate was significantly lower in the miR-320a mimics group than in the mimic-NC group, while the miR-320a inhibitor group had a higher apoptosis rate compared to the inhibitor-NC group (Fig. 2C and Fig. S1). This indicates that miR-320a overexpression may suppress cellular apoptosis, whereas its knockdown may enhance this process.

There was an interaction between miR-320a and PIK3CD

Target genes of miR-320a were predicted using the miRDB database, and GDM-related genes were retrieved from the GeneCards database with a screening criterion of a relevance score of 65 and above. The intersection of the two gene datasets was then obtained, yielding a total of 11 common genes (Fig. 3A). We ultimately selected PIK3CD for subsequent investigations, primarily due to its involvement in the insulin signaling pathway and inflammatory responses [20, 21], which are closely associated with the pathogenesis and progression of GDM. In addition, bioinformatics prediction revealed potential binding sites between miR-320a and PIK3CD 3’-UTR (Fig. 3B), further supporting our selection of this gene for follow-up functional validation. Studies have demonstrated that PIK3CD is highly expressed in the serum of GDM patients, and PIK3CD mRNA in leukocytes is significantly positively correlated with plasma glucose concentration at the 2-hour point of the 75 g OGTT, as well as SIRT1 mRNA [16]. Further validation experiments demonstrated that both mRNA and protein expression levels of PIK3CD were significantly elevated in HG‑stimulated HTR‑8/SVneo cells compared with control cells (Fig. 3C-D). The full uncropped Western blot membrane corresponding to Fig. 3D was placed in Supplementary Fig. S3-1. Dual‑luciferase reporter assays showed that transfection with miR‑320a mimics markedly reduced luciferase activity in cells carrying PIK3CD‑WT compared with NC mimics, supporting a direct interaction between miR‑320a and PIK3CD. No significant changes in luciferase activity were detected in cells transfected with PIK3CD‑MUT. Consistent with this observation, miR‑320a inhibitor transfection enhanced luciferase activity in the PIK3CD‑WT group but not in the PIK3CD‑MUT group (Fig. 3E). Moreover, overexpression of miR‑320a significantly downregulated the expression of PIK3CD at the mRNA and protein levels (Fig. 3F-G). The full uncropped Western blot membrane corresponding to Fig. 3G was placed in Supplementary Figs. S3-2.

Fig. 3.

Relationship between miR-320a and PIK3CD. A Venn diagram to search for miR-320a downstream target genes. B Bioinformatic prediction of the binding site between miR-320a and the 3’-UTR of PIK3CD. C PIK3CD mRNA expression in HTR-8/SVneo cells under normal isotonic control and high-glucose (HG) culture conditions. D PIK3CD protein expression in HTR-8/SVneo cells under normal isotonic control and high-glucose (HG) culture conditions. E Dual-luciferase reporter assay to detect the interaction between PIK3CD and miR-320a. F Effect of miR-320a on PIK3CD mRNA expression level. G Effect of miR-320a on PIK3CD protein expression level. ***p < 0.001

MiR-320a affects the progression of GDM cells by regulating PIK3CD

To investigate the functional role of miR‑320a via targeting PIK3CD in HTR‑8/SVneo cells, efficient regulation of the target gene was first verified. Transfection with miR‑320a mimics significantly decreased both mRNA and protein expression of PIK3CD compared with mimics‑NC. By contrast, co‑transfection of miR‑320a mimics with the PIK3CD overexpression plasmid (pcDNA‑PIK3CD) effectively restored PIK3CD mRNA and protein expression (Fig. 4A-B), confirming successful transfection and targeted gene regulation. The full uncropped Western blot membrane corresponding to Fig. 4B was placed in Supplementary Fig. S3-3.

Fig. 4.

PIK3CD-mediated regulation of miR-320a effects on HTR-8/SVneo cell proliferation and apoptosis. A PIK3CD mRNA expression in HTR-8/SVneo cells. B PIK3CD protein expression in HTR-8/SVneo cells. C CCK-8 assay evaluating proliferation under miR-320a modulation targeting PIK3CD. D Flow cytometric analysis of apoptosis following miR-320a manipulation via PIK3CD regulation. ***p < 0.001

Subsequent functional assays demonstrated that miR‑320a overexpression significantly enhanced the proliferation of HTR‑8/SVneo cells, as indicated by increased cell viability relative to the mimics‑NC group. This pro‑proliferative effect was significantly reversed by co‑expression of pcDNA‑PIK3CD (Fig. 4C). Furthermore, transfection with miR‑320a mimics markedly reduced the apoptotic rate compared with the mimics‑NC group, and this anti‑apoptotic effect was significantly attenuated by co‑transfection with pcDNA‑PIK3CD (Fig. 4D and Fig. S2).

Collectively, these data indicate that miR‑320a promotes proliferation and inhibits apoptosis in HTR‑8/SVneo cells, and these effects can be partially reversed by upregulation of PIK3CD.

Discussion

As obesity rates rise globally, more pregnant women are being diagnosed with GDM. These pregnant women are at increased risk of multiple pregnancy complications, including fetal macrosomia, preterm delivery, and operative interventions, including cesarean Sect [22]. GDM diagnosis via OGTT is commonly performed in early or mid-to-late pregnancy. The OGTT, despite being the primary diagnostic method, has several drawbacks, including issues related to biological variability, testing requirements, diagnostic standards, and timing. These problems can result in undiagnosed, misdiagnosed, and untreated GDM cases, posing potential risks to pregnant women and fetuses [3]. Furthermore, although emerging oral hypoglycemic drugs like glyburide and metformin show promise, their long-term safety for mothers and children remains a concern [23]. These challenges underscore the need for novel biomarkers to facilitate earlier GDM detection and intervention, ultimately leading to improved pregnancy outcomes.

Research increasingly implicates miRNAs in the pathogenesis of diverse human diseases, including cancer, autoimmune diseases, and cardiovascular diseases. In the field of GDM, miRNAs also play a significant role. Emerging evidence underscores the significant involvement of miRNAs in diabetes pathogenesis and its complications, highlighting their potential as diagnostic and prognostic biomarkers. Specifically, downregulation of miR-96-5p has been observed in the placental tissue of GDM patients, suggesting its role in GDM pathology [24]. Furthermore, miR-335-5p modulates insulin resistance and pancreatic β-cell function in GDM mouse models [25]. Beyond GDM, circulating miR-320a has been widely reported in diabetic complications. Decreased plasma levels of miR-320a are associated with diabetic retinopathy (DR) in patients compared to healthy controls, implicating this miRNA in DR development [26]. Additionally, regarding microvascular complications, research in diabetic nephropathy identifies miR-320a as a key aggravator of renal injury and positions it as a promising therapeutic focus for this condition [14].

Building upon this foundation, the present study investigated the expression and functional role of miR-320a in GDM. Our results showed a consistent downregulation of miR-320a in clinical GDM serum samples and in an in vitro HG-induced cell model. ROC curve analysis demonstrated that miR-320a effectively distinguished GDM patients from healthy controls, and binary logistic regression analysis revealed a significant correlation between low miR-320a expression and an increased risk of fetal macrosomia. Furthermore, in vitro, HG-treated HTR-8/SVneo cells exhibited increased viability and decreased apoptosis following miR-320a overexpression, contrasting with effects observed upon miR-320a suppression. These results indicate that miR-320a may protect placental cells from HG-induced dysfunction and thus reduce the risk of adverse pregnancy outcomes. Mechanistically, bioinformatics analysis and dual-luciferase assays confirmed that PIK3CD was a direct and functional downstream target of miR-320a, and PIK3CD expression was elevated under HG conditions. PIK3CD is closely involved in insulin signaling and inflammatory responses, which are closely associated with the pathogenesis and progression of GDM [20]. MiR-320a overexpression suppressed PIK3CD expression, while co-transfection with pcDNA-PIK3CD significantly reversed miR-320a-mediated effects on cell viability and apoptosis. Our data therefore suggest that miR-320a exerts its regulatory effect in GDM progression by modulating the expression level of PIK3CD.

These findings collectively suggested that miR-320a may exert tissue-specific effects: detrimental in renal tissue, yet potentially protective in the placenta and retina. Several factors may explain this discrepancy. First, the cellular origin of circulating miR-320a may differ among different diabetic conditions: in GDM, placental trophoblasts may be the primary source, whereas in diabetic nephropathy, renal tubular cells predominate. Second, the directionality of miR-320a change (up- versus downregulation) may reflect distinct pathophysiological phases: our GDM cohort represents a gestational, potentially reversible metabolic stress state, whereas prior studies in diabetic nephropathy typically involve chronic, established disease. Collectively, these findings position miRNAs as promising candidates for early detection and therapeutic strategies in GDM and related diabetic conditions.

Notably, this study has several strengths that support the reliability of the present findings. First, the research design integrated both clinical and in vitro experimental evidence, combining serum samples from 40 GDM patients and 40 age- and gestational age-matched healthy controls with functional experiments on HTR-8/SVneo cells stimulated by HG (25 mmol/L), which effectively enhanced the reliability and translational value of the findings. Second, the study systematically explored the role of miR-320a in GDM from multiple dimensions, including its diagnostic potential for GDM (evaluated by ROC curve analysis), predictive value for fetal macrosomia (verified by binary logistic regression), and regulatory effects on cell proliferation and apoptosis, as well as its underlying molecular mechanism via targeting PIK3CD. Third, the identification of the miR-320a/PIK3CD axis provided a novel and specific molecular target for the diagnosis and potential treatment of GDM, which enriched the existing understanding of the epigenetic regulation mechanisms in GDM pathogenesis and offered new insights for clinical practice.

However, several limitations should be considered. This study had a modest sample size, single-center enrollment, and a cross-sectional design, which may limit the generalizability of the findings. The relatively low events-per-variable ratio (approximately 2.71) in the multivariable logistic regression model might compromise the stability of effect estimates. Future large-scale, multicenter prospective cohorts are needed to validate the predictive efficacy of miR-320a in broader populations, optimize multivariate model stability, and allow more comprehensive adjustment for confounding factors, including longitudinal glycemic control and gestational weight gain trajectories. In addition, we only verified the stability of U6 by calculating the standard deviation of raw Ct values, and further stability assessment of U6 via geNorm or NormFinder is required in subsequent research. All in vitro functional observations rely solely on the HTR-8/SVneo cell line, and the miR-320a/PIK3CD regulatory axis has not been verified in animal models or other in vivo systems. Although in vivo validation is currently limited by cost and technical constraints, further animal experiments are necessary to confirm the biological role of this axis in GDM pathogenesis. This study only verified the regulatory relationship between miR-320a and PIK3CD at the protein level, without detecting phosphorylated AKT or S6 to confirm downstream PI3K pathway activation. Subsequent work will supplement p-AKT and p-S6 measurement to further clarify the involvement of the miR-320a/PIK3CD axis in PI3K signaling. Notably, an inconsistency was observed between clinical and in vitro results: decreased serum miR-320a correlated with fetal macrosomia in GDM patients, whereas miR-320a overexpression promoted trophoblast proliferation and inhibited apoptosis in HTR-8/SVneo cells. This discrepancy may stem from the difference between circulating systemic miR-320a and its local function in placental tissues, cell-type-specific regulatory patterns, and mismatched gestational stages between the cell line and clinical samples. The exact mechanism underlying this inconsistency remains unclear, requiring further well-designed clinical and experimental research. The diagnostic and therapeutic potential of miR-320a for GDM still requires rigorous external validation in independent clinical cohorts and preclinical models.

Conclusion

In summary, miR-320a was significantly downregulated in the serum of GDM patients and may serve as a promising diagnostic and therapeutic candidate biomarker for GDM. Overexpression of miR-320a promoted the proliferation and inhibited the apoptosis of HTR-8/SVneo cells, and it exerted this regulatory effect on HTR-8/SVneo cell biological behaviors by directly targeting PIK3CD.

Authors’ contributions

All authors have given substantial contributions to the conception or the design of the manuscript, RP Y, XY L and S Z to acquisition, analysis and interpretation of the data. RP Y, XY L have participated to drafting the manuscript, S Z revised it critically. All authors read and approved the final version of the manuscript.

Funding

The authors declare that no funds, grants, or other support was received during the preparation of this manuscript.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

Approval for this study was granted by the Ethics Committee of Shijiazhuang Maternity & Child Healthcare Hospital. All procedures involving human participants were conducted in accordance with the Declaration of Helsinki and its subsequent amendments or comparable ethical standards. Written informed consent was obtained from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.