Abstract

   Pediatric non-alcoholic fatty liver disease (NAFLD) is emerging as a
   worldwide health concern with the potential to advance to cirrhosis and
   liver cancer. NAFLD can also directly contribute to heart problems
   through inflammation and insulin resistance, even in individuals
   without other risk factors. The pathological mechanisms of NAFLD are
   linked to functional differences of miRNAs in different biological
   environments. The miRNA in serum exosomes may reflect the pathological
   state of the liver and changes in systemic metabolism, while the miRNA
   in serum may be associated with physiological processes other than the
   liver. Pediatric non-alcoholic fatty liver disease (NAFLD) is emerging
   as a worldwide health concern with the potential to advance to
   cirrhosis and liver cancer. NAFLD can also directly contribute to heart
   problems through inflammation and insulin resistance, even in
   individuals without other risk factors. The pathological mechanisms of
   NAFLD are linked to functional differences of miRNAs in different
   biological environments. The miRNA in serum exosomes may reflect the
   pathological state of the liver and changes in systemic metabolism,
   while the miRNA in serum may be associated with physiological processes
   other than the liver. Pediatric non-alcoholic fatty liver disease
   (NAFLD) is emerging as a worldwide health concern with the potential to
   advance to cirrhosis and liver cancer. NAFLD can also directly
   contribute to heart problems through inflammation and insulin
   resistance, even in individuals without other risk factors. The
   pathological mechanisms of NAFLD are linked to functional differences
   of miRNAs in different biological environments. The miRNA in serum
   exosomes may reflect the pathological state of the liver and changes in
   systemic metabolism, while the miRNA in serum may be associated with
   physiological processes other than the liver. Our study identified 36
   miRNAs with differential expression in the serum of NAFLD patients
   compared to the control group, including 21 miRNAs with significantly
   increased expression and 15 with decreased expression. Consistent with
   our previously reported data on serum-derived exosomal miRNA profiling,
   this study also observed a notable upregulation of serum miR-122-5p
   levels in NAFLD patients. PCR validation confirmed the differential
   expression of miR-122-5p identified through RNA sequencing. Functional
   analysis using GO and KEGG pathways revealed a diverse range of
   biological roles associated with these differentially expressed miRNAs.
   Notably, NAFLD significantly impacts heart health, with miR-122-5p
   playing a key role in regulating cardiovascular function. Furthermore,
   activation of the miR-122/Sirt-6/ACE2 axis may contribute to myocardial
   necrosis, highlighting its potential role in NAFLD-associated
   cardiovascular risks. Our study suggests that miR-122 plays a key role
   in the progression of NAFLD and its associated metabolic disturbances,
   which can increase the risk of cardiovascular disease. Targeting
   miR-122 may offer potential therapeutic benefits for improving both
   liver and heart health in individuals with NAFLD.

   Keywords: NAFLD, Serum, miRNAs, miR-122-5p, Cardiovascular

   Subject terms: Biomarkers, Diseases, Medical research, Molecular
   medicine, Pathogenesis

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
   NAFLD^[30]1. 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
   process^[31]2,[32]3. Serum exosome, a nano-sized extracellular vesicle
   with an endocytic origin, also contains a variety of proteins and
   miRNAs that mediate intercellular communication^[33]4. 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
   group^[34]5. There is a significant association between NAFLD and
   cardiovascular disease (CVD), especially in the more severe forms of
   NAFLD^[35]6. This association may be related to metabolic abnormalities
   in patients with NAFLD, including insulin resistance, lipid metabolism
   disorders, and inflammatory responses^[36]7. 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 disease^[37]8. 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
   circulation^[38]6.

   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 severity^[39]9,[40]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 previously^[41]11. 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 [42]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 [43]1, while the ultrasound
   examination results are shown in Figure [44]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.
   Reference range NAFLD No-NAFLD p
   n 3 3
   Age (years) 12. 1 ± 0. 53 11. 9 ± 1. 79 0. 846
   Hight(cm) 155. 7 ± 10. 02 156. 2 ± 5. 74 0. 94
   Weight(Kg) 77. 0 ± 15. 87 69. 33 ± 3. 06 0. 477
   BMI(Kg/m2) 31. 5 ± 3. 06 28. 38 ± 0. 75 0. 158
   Waist circumference(cm) 99. 0 ± 9. 54 87. 67 ± 2. 52 0. 117
   Hips circumference(cm) 101. 3 ± 8. 08 100. 33 ± 0. 58 0. 841
   SBP 122. 3 ± 30. 4 115. 6 ± 4. 6 0. 700
   DBP 65. 3 ± 11. 6 62. 6 ± 6. 1 0. 742
   AST((U/L) < 60 112. 3 ± 71. 45 18. 3 ± 3. 51 0. 085
   ALT((U/L) < 50 61. 3 ± 33. 95 23. 0 ± 4. 58 0. 125
   TG (mmol/L) < 1. 7 1. 25 ± 0. 10 1. 19 ± 0. 65 0. 876
   CHO (mmol/L) < 5. 7 5. 16 ± 0. 71 4. 62 ± 1. 09 0. 508
   HLD-C (mmol/L) > 1. 04 1. 23 ± 0. 04 1. 35 ± 0. 07 0. 058
   UA 372. 00 ± 72. 19 384. 33 ± 114. 12 0. 882
   Apo A 61. 33 ± 10. 69 114. 00 ± 44. 98 0. 120
   FBG 5. 37 ± 0. 21 5. 43 ± 0. 25 0. 742
   INS 22. 17 ± 7. 93 15. 33 ± 5. 87 0. 297
   HOMA-IR 5. 34 ± 2. 13 3. 66 ± 1. 24 0. 304
   [45]Open in a new tab

   BMI body mass index, SBP Systolic blood pressure, DBP Diastolic blood
   pressure, UA Uric acid, Apo A Apolipoprotein A, TG triglyceride, CHO
   cholesterol, HLD-C high density lipoprotein cholesterol, LDL-C low
   density lipoprotein cholesterol, FBG Fasting blood glucose, INS Fasting
   insulin.

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 [46]2. We
   discovered 1523 miRNAs by combining conserved and new miRNAs
   (Supplemental Table [47]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 [48]S2).

Table 2.

   Showing the results of non-coding RNA sequencing data mapping to the
   human genomes.
   NAFLD1 NAFLD2 NAFLD3 NON-NAFLD1 NON-NAFLD2 NON-NAFLD3
   Total reads 12,407,505 7,719,278 6,588,206 11,288,361 10,284,175
   7,431,616
   Mapped reads 10,591,935 6,916,761 5,190,927 10,577,766 8,705,516
   7,005,686
   Mapped rate 85. 37% 89. 60% 78. 79% 93. 71% 84. 65% 94. 27%
   +’ Mapped reads 6,873,301 4,114,777 3,397,171 6,824,674 5,925,342
   4,924,836
   +’ Mapped reads rate 55. 40% 53. 31% 51. 56% 60. 46% 57. 62% 66. 27%
   -’ Mapped reads 3,718,634 2,801,984 1,793,756 3,753,092 2,780,174
   2,080,850
   -’ Mapped reads rate 29. 97% 36. 30% 27. 23% 33. 25% 27. 03% 28. 00%
   [49]Open in a new tab

   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. [50]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. [51]2).

Fig. 1.

   [52]Fig. 1
   [53]Open in a new tab

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

Fig. 2.

   [54]Fig. 2
   [55]Open in a new tab

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

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. [56]3). Enrichment
   analysis using KEGG pathways was conducted on miRNA targets that were
   differentially expressed to identify the top 20 enriched terms
   (Fig. [57]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.

   [58]Fig. 3
   [59]Open in a new tab

   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.

Fig. 4.

   [60]Fig. 4
   [61]Open in a new tab

   Top 20 Statistics of Pathway Enrichment.

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
   time^[62]12. 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
   buildup^[63]13,[64]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 group^[65]5. 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 NAFLD^[66]15.
   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
   pathway^[67]16. 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 metabolism^[68]17. 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 disease^[69]18. 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 analysis^[70]19,[71]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
   conditions^[72]21.

   NAFLD has a notable impact on heart health, with miR-122-5p playing a
   key role in regulating lipid metabolism, inflammation, and endothelial
   function^[73]22,[74]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 function^[75]24,[76]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
   infarction^[77]26. 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 NAFLD^[78]27.

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.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [79]Supplementary Material 1^ (136.6KB, xlsx)
   [80]Supplementary Material 2^ (12.8KB, xlsx)
   [81]Supplementary Material 3^ (2.6MB, pdf)
   [82]Supplementary Material 4^ (1.8MB, pdf)

Author contributions

   Jian-Wei Zhang and Hai-Tao Pan performed experiments. Jian-Wei Zhang,
   Kamran Ullah, and Hai-Tao Pan wrote the main manuscript text. Jian-Wei
   Zhang and Nauman Khan prepared Figs. 1, 2, 3 and 4. Jian-Wei Zhang and
   Kamran Ullah prepared Table 1, and 2. All authors reviewed the
   manuscript.

Funding

   This work was supported by the National Natural Science Foundation of
   China (82071729), the Science Technology Department of Zhejiang
   Province, China (LTGY23H040004, LTGY23H040005, LTGY24H040006), the
   Health Commission of Zhejiang Province, China (2022KY1306), the Health
   Commission of Shaoxing, China (2022KY040, 2023SKY047), the Science
   Technology Department of Shaoxing (2022A14006, 2023A14032).

Data availability

   Sequence data that support the findings of this study can be accessed
   through the NCBI SRA resource: PRJNA821449.

Declarations

Competing interests

   The authors declare no competing interests.

Informed consent

   For research involving human participants who are minors, including
   tissue sample donors, informed consent was obtained from a parent
   and/or legal guardian.

Ethical approval

   This study received approval from the Shaoxing Maternal and Child
   Health Care Hospital’s Ethics Committee. Everyone who participated in
   the study gave informed consent.

Footnotes

   Publisher’s note

   Springer Nature remains neutral with regard to jurisdictional claims in
   published maps and institutional affiliations.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-87791-1.

References