Introduction

Childhood non-alcoholic fatty liver disease (NAFLD) is emerging as a leading contributor to long-term liver conditions linked to a significant overhaul of liver metabolism. Previously reported data demonstrated that deregulation of the expressions and activities of microRNAs (miRNAs) have a key role in metabolic disorders linked with NAFLD1. miRNAs are short endogenous non-coding RNA composed of about 20–30 nucleotides, which regulate about 90% of human genes and have a significant effect on gene expression in almost every biological process2,3. Serum exosome, a nano-sized extracellular vesicle with an endocytic origin, also contains a variety of proteins and miRNAs that mediate intercellular communication4. In our 2022 study, we demonstrated a marked variation in the expression of 80 miRNAs, including miR-122-5p, miR-27a, and miR‐335-5p, in exosomes derived from serum of children with NAFLD compared to the control group5. There is a significant association between NAFLD and cardiovascular disease (CVD), especially in the more severe forms of NAFLD6. This association may be related to metabolic abnormalities in patients with NAFLD, including insulin resistance, lipid metabolism disorders, and inflammatory responses7. In patients with NAFLD, the elevation of miR-122-5 may be associated with fat accumulation and inflammatory response in the liver, all of which may contribute to the development of cardiovascular disease8. In addition, the increased risk of cardiovascular events in patients with NAFLD may be related to the release of inflammatory factors and metabolites from their liver, which may affect the cardiovascular system through blood circulation6.

Circulating miRNAs, along with exosomal miRNAs, have garnered significant attention as a possible diagnostic biomarker for clinical use. Deregulation in circulatory miRNA expressions causes a variety of chronic illnesses, including chronic liver disease. miRNA-34a was up-regulated in serum samples from patients with NAFLD, and its levels of expression were linked with disease severity9,10. Our goal in this study was to create a detailed microRNA profile of serum samples from individuals with non-alcoholic fatty liver disease (NAFLD) and explore how different microRNAs could affect cardiovascular well-being.

Materials and methods

Sample Collection and RNA extraction

The local Ethics Committee approved the ethics application (Ethics Committee of Shaoxing Maternal and Child Health Care Hospital, NO. 2018035). All participants provided written informed consent in accordance with the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. Between July and December 2019, the Pediatric Endocrinology Department at our hospital selected three children with non-alcoholic fatty liver disease who were admitted to the hospital and matched them with three other patients for comparison purposes. Detailed inclusion and exclusion criteria have been published previously11. At the Shaoxing Maternal and Child Health Care Hospital, every patient underwent medical check-ups to ensure their well-being. Serum was extracted from 3 mL of blood for RNA extraction.

RNA library preparation and sequencing

To create the small RNA library, 3 µg of total RNA was isolated for each sample. Following the instructions provided by the manufacturer, libraries for sequencing were generated utilizing the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA), with unique index codes assigned to each sample for sequence identification. The samples indexed were grouped together using a cBot Cluster Generation System with TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the instructions provided by the manufacturer. The library samples were examined using an Illumina Hiseq 2500/2000 system, resulting in the generation of 50 bp single-end reads following the formation of clusters.

The quality of the sequencing data was evaluated using Fast-QC software from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, which analyzed minimum quality standards, high-value region dispersion, GC content, PCR duplication content, and Kmer frequency. The DEGseq (2010) R package was utilized to analyze three samples for variations in expression. q-value was used to alter the P-value. P-value < 0. 01 and log2(fold change) > 1 were defined as the default significance threshold for variable expression Levels.

miRNA target genes were predicted using TargetScan, miRanda, and miRDB. DAVID was utilized for Annotation, Visualization, and Integrated Discovery to conduct GO enrichment and KEGG pathway enrichment analysis.

qPCR analysis

Extraction of miRNAs from serum was performed using a miRcute miRNA isolation kit from TIANGEN in Beijing, China. The miR-122-5p forward primer sequence is 5′-TATTCGCACTGGATACGACACAAAC-3′, while the reverse primer sequence is 5′-GCCCGTGGAGTGTGACAATGGT-3′. The RT-qPCR was conducted simultaneously using the StepOne Plus Real-Time PCR System from Applied Biosystems and the SYBR Green PCR master mix from Tiangen Biotechnology Co., Ltd. Beijing-based company Tiangen provided all miRNA primers.

Statistical analysis

Student t-test was utilized to analyse data from real-time qPCR. Data are presented as the means ± standard deviations (SD). Statistical analysis was performed using the hypergeometric distribution method; the most significant function was calculated, and P < 0. 05 was used as the threshold of significant gene enrichment.

Results

Characteristics of patients

Patients with obesity and NAFLD (n = 3) had a mean age of 12.1 ± 0.53 years and a BMI of 31.5 ± 3.06. In contrast, controls with obesity but without NAFLD had a mean age of 11.9 ± 1.79 years and a BMI of 28.38 ± 0.75. Age, weight, height, BMI, and triglyceride (TG) did not differ significantly between the groups. Demographic and clinical data for each subject are presented in Table 1, while the ultrasound examination results are shown in Figure S1. The results suggest enhanced liver echo, abnormal attenuation of ultrasound signals (i.e. poor display of deep liver and diaphragm), and loss of normal liver echo texture (including poor display of blood vessel wall).

Table 1 The characteristics of patients with NAFLD and healthy controls.
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Analysis of NAFLD serum microRNAs

Before analyzing the raw sequencing data for quality control, it is possible to undertake a cursory inspection of the data. RNA sequencing and evaluation were performed on three serum samples from individuals with NAFLD and three samples from individuals without NAFLD. NAFLD patients’ serum contained an average of 80. 1 million valid reads, while healthy controls’ serum had 87. 0 million valid reads on average. Most of the accurate interpretations from the NAFLD and NON-NAFLD serum samples were effectively linked to the human non-coding RNA dataset, with percentages of 84. 6% and 90. 0% shown in Table 2. We discovered 1523 miRNAs by combining conserved and new miRNAs (Supplemental Table S1). A notable contrast was observed in serum miRNA concentrations between the NAFLD and control groups (P < 0. 05), showing an increase in 21 miRNAs and a decrease in 15 miRNAs (Supplemental Table S2).

Table 2 Showing the results of non-coding RNA sequencing data mapping to the human genomes.
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The differential miRNA expression in NAFLD versus non-NAFLD samples was then investigated using hierarchical cluster analysis to construct a heat map evaluating the outcomes of the two groups as illustrated in Fig. 1. Based on the comparison between NAFLD and controls, we selected significantly 10 up-regulated microRNAs (hsa-miR-122-5p, hsa-miR-378d, hsa-miR-885-5p, hsa-miR-4686, hsa-miR-144-5p, hsa-miR-4485-3p, hsa-miR-122-3p, hsa-miR-885-3p, hsa-miR-15a-5p, and hsa-miR-192-3p) and 10 downregulated microRNAs (hsa-miR-206, hsa-miR-873-5p, hsa-miR-873-3p, hsa-miR-664b-5p, hsa-miR-409-5p, hsa-miR-1291, hsa-miR-195-3p, hsa-miR-370-3p, hsa-miR-370-3p, and hsa-miR-1246) for further investigation. miR-122-5p was detected in the serum of individuals with both NAFLD and non-NAFLD (Fig. 2).

Fig. 1
figure 1

Cluster analysis of differentially expressed serum miRNAs. Heat map of differentially expressed microRNAs from NAFLD (n = 3) and normal controls (n = 3).

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Fig. 2
figure 2

Validation of miR-122-5p by qRT-PCR(*: P < 0. 05).

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Target genes predicted and their KEGG and GO annotations

Using target genes, the roles of these miRNAs with varying expression levels were hypothesized. miRanda was employed to forecast the target gene of microRNAs. Eighty miRNAs with differential expression were hypothesized to target 36,718 mRNAs. The GO analysis revealed the top 20 hits for biological process (BP), cellular component (CC), and molecular function (MF) in the presentation (Fig. 3). Enrichment analysis using KEGG pathways was conducted on miRNA targets that were differentially expressed to identify the top 20 enriched terms (Fig. 4). The KEGG pathway enrichment analysis shows that the miRNAs with differential expression are primarily associated with the vascular smooth muscle contraction, ubiquitin-mediated proteolysis, transcriptional misregulation in cancer and toll-like receptor signaling pathway.

Fig. 3
figure 3

Gene ontology (GO) enrichment analysis of predicted target genes. GO enrichment analysis according to three GO categories of biological process (BP), molecular functions (MF), and cellular component (CC) was performed on the differentially expressed genes.

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Fig. 4
figure 4

Top 20 Statistics of Pathway Enrichment.

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Discussion

Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the most prevalent chronic conditions among children. The early onset of NAFLD poses a significant risk, as prolonged exposure to the disease and its metabolic complications can lead to severe health outcomes over time12. This highlights the critical need for early diagnosis and timely intervention to reduce long-term risks and improve outcomes in children with NAFLD. In recent times, there has been an increased focus on the role of miRNA in regulating genes related to liver health and diseases. Earlier studies have indicated that miRNAs dysregulated networks play a part in the development of NAFLD, including factors like oxidative stress, hepatic inflammation, and lipid buildup13,14. By employing high-throughput sequencing methods, we discovered 36 miRNAs with varying levels in the serum of children with NAFLD compared to the control group. Our previous data indicated a notable variation of 80 exosomal miRNAs, with 30 showing an increase and 50 showing a decrease in the same group of children compared to the control group5. Consistent with our earlier findings, we demonstrated a novel association between elevated serum miR-122-5p levels and competition with serum exosomal microRNAs in pediatric NAFLD patients, suggesting a potential role in predicting disease advancement. Our findings using real-time PCR indicate that there are consistent differences in serum miR-122-5p expression levels between children with NAFLD and control subjects, suggesting that it could serve as a more sensitive diagnostic tool for NAFLD. Our discovery regarding miR-122-5p aligns with a recent study that conducted miRNA sequencing on plasma samples from individuals with NAFLD15. Furthermore, we identified variations in the expression levels of several crucial miRNAs, including 10 up-regulated microRNAs (hsa-miR-122-5p, hsa-miR-378d, hsa-miR-885-5p, hsa-miR-4686, hsa-miR-144-5p, hsa-miR-4485-3p, hsa-miR-122-3p, hsa-miR-885-3p, hsa-miR-15a-5p, and hsa-miR-192-3p) and 10 significantly downregulated microRNAs (hsa-miR-206, hsa-miR-873-5p, hsa-miR-873-3p, hsa-miR-664b-5p, hsa-miR-409-5p, hsa-miR-1291, hsa-miR-195-3p, hsa-miR-370-3p, hsa-miR-370-3p, and hsa-miR-1246) in the comparison between the NAFLD and control groups. We also pick the above miRNAs to construct a heat map, using hierarchical cluster analysis. Heat maps clearly showed differences in miRNA expression between NAFLD and control groups. According to our recent research, previous studies have shown that miR-378 is crucial in the progression of liver inflammation and fibrosis through its positive control of the NF-κB-TNFα pathway16. In the same way, blood microRNA analysis indicated a direct correlation between levels of hsa-miR-122-5p and hsa-miR-885-5p with fatty liver and associated lipoprotein metabolism17. In addition, the levels of hepatic miR-122-3p, miR-140-5p, and miR-148b-5p are associated with serum cytokeratin-18 levels in metabolic-associated fatty liver disease18. The present research revealed enrichment of the vascular smooth muscle contraction, ubiquitin-mediated proteolysis, transcriptional misregulation in cancer, and toll-like receptor signaling pathway through KEGG analysis. Newly released information shows that toll-like receptor signaling exacerbates alcoholic liver disease and liver damage in non-alcoholic steatohepatitis, which aligns with the findings of KEGG pathway enrichment analysis19,20. Likewise, strong evidence has shown that the hedgehog pathway’s imbalance plays a crucial role in the progression of hepatic steatosis and the transition from hepatic steatosis to more severe liver conditions21.

NAFLD has a notable impact on heart health, with miR-122-5p playing a key role in regulating lipid metabolism, inflammation, and endothelial function22,23. In our study, apolipoprotein A levels in the NAFLD group were slightly lower, and common carotid artery thickness was marginally higher compared to the non-NAFLD group. Although these differences were not statistically significant, likely due to the small sample size, a trend of change was evident. These findings highlight potential distinctions between the groups and suggest that elevated miR-122-5p expression could increase the risk of cardiovascular disease in children with NAFLD. High levels of miR-122-5p are linked to higher lipid buildup and changes in inflammatory reactions, which play a role in the advancement of atherosclerosis and negative changes in heart structure. The significance of this microRNA in preserving vascular integrity and reducing cardiovascular complications is highlighted by its impact on endothelial function24,25. Notably, miR-122 targets the sirtuin-6 (Sirt-6) gene, which is an essential regulator of cardiovascular function and is believed to be a partial angiotensin-converting enzyme 2(ACE2) activator. Modulation of this axis is thought to contribute to the pathogenesis of myocardial infarction26. New research has emphasized the various ways it affects cardiovascular disease, indicating that miR-122-5p could be a valuable tool for diagnosing and treating cardiovascular conditions associated with NAFLD27.

Conclusion

In summary, the miRNA expression profiles in the serum of children with NAFLD are markedly different from those in the control group. The cognition of these miRNAs can help to identify key factors and risks for disease prognosis and treatment. Additionally, our research revealed an increase in serum miR-122-5p levels in children diagnosed with NAFLD, aligning with our prior findings on serum exosomal microRNAs in this group of patients. In addition, our research emphasizes the important regulatory function of miR-122-5p in maintaining cardiovascular well-being, connecting NAFLD to cardiovascular issues. Our results may also provide mechanistic insights into the development of cardiovascular diseases in NAFLD patients.