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The significance of m6A RNA methylation regulators in diagnosis and subtype classification of HBV-related hepatocellular carcinoma

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Abstract

In recent years, abnormal m6A alteration in hepatocellular carcinoma (HCC) has been a focus on investigating the biological implications. In this study, our objective is to determine whether m6A modification contributes to the progression of HBV-related HCC. To achieve this, we employed a random forest model to screen top 8 characteristic m6A regulators from 19 candidate genes. Subsequently, we developed a nomogram model that utilizes these 8 characteristic m6A regulators to predict the prevalence of HBV-related HCC. According to decision curve analysis, patients may benefit from the nomogram model. The clinical impact curves exhibited a robust predictive capability of the nomogram models. Additionally, consensus molecular subtyping was employed to identify m6A modification patterns and m6A-related gene signature. The quantification of immune cell subsets was accomplished through the implementation of ssGSEA algorithms. PCA algorithms were developed to compute the m6A score for individual tumors. Two distinct m6A modification patterns, namely cluster A and cluster B, exhibited significant correlations with distinct immune infiltration patterns and biological pathways. Notably, patients belonging to cluster B demonstrated higher m6A scores compared to those in cluster A, as determined by the m6A score metric. Furthermore, the expression of IGFBP3 proteins was validated through immunofluorescence, revealing their pronounced lower expression in tumor tissues. In summary, our study underscores the importance of m6A modification in the advancement of HBV-related HCC. This research has the potential to yield novel prognostic biomarkers and therapeutic targets for the identification of HBV-related HCC.

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Data availability

The RNAseq data that support the findings of this study are openly available in GEO database at http://www.ncbi.nlm.nih.gov/geo/. (Accession number: GSE121248 and GSE22058). The liver tissue data are not publicly available due to ethical restriction.

Abbreviations

m6A:

N6-methyladenosine

HCC:

Hepatocellular carcinomas

HBV:

Hepatitis B virus

HBV-related HCC:

Hepatitis B virus-related hepatocellular carcinomas

cccDNA:

Covalently closed circular DNA

GEO:

Gene Expression Omnibus

RF:

Random forest

SVM:

Support vector machine

ROC:

Receiver operating characteristic

DCA:

Decision curve analysis

ssGSEA:

Single sample gene set enrichment analysis

MDS:

Multidimensional scaling

DEGs:

Differentially expressed genes

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

DAPI:

4′,6-Diamidino-2-phenylindole

CHB:

Chronic hepatitis B

IGFBP3:

Insulin-like growth factor binding protein 3

IGFBPs:

Insulin‐like growth factor binding proteins

IGF1:

Insulin‐like growth factor‐1

HNRNPC:

Heterogeneous nuclear ribonucleoprotein C

RBM15:

RNA-binding motif protein 15

YTHDF2:

YTH N6-Methyladenosine RNA Binding Protein F2

WTAP:

Wilms tumor 1-associated protein

METTL3:

Methyltransferase-like 3

METTL14:

Methyltransferase-like 14

pri-miR126:

Primary miR126

KCs:

Kupffer cells

TNF:

Tumor necrosis factor

JNK:

JUN N-terminal kinases

VEGFs:

Vascular endothelial growth factor

CXCL1:

Chemokine (C–X–C motif) ligand 1

CXCL8:

Chemokine (C–X–C motif) ligand 8

CCL1:

Chemokine ligand 1

CCL5:

Chemokine ligand 5

IL-6:

Interleukin-6

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Acknowledgements

We appreciate contributors for sharing their valuable datasets and GEO Database for giving their platforms. Then, we would like to thank the participating patients for the source of HBV-related HCC tissue specimens.

Funding

This research was funded by Natural Science Foundation of China and Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University, China. Grant/Award Number: 82070641, 81971310 and 2022-XKCRC-04.

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Authors and Affiliations

  1. Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, No. 277 Yanta Road(W), Xi’an, 710061, Shaanxi, China

    Qijuan Zang, Siyi Liu, Chengbin Zhu, Liangru Liu, Weicheng Xu & Yingli He

  2. Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi, China

    Yalin Ju & Shaobo Wu

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  1. Qijuan Zang
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Contributions

Qijuan Zang and Yalin Ju have the same contribution. Conceptualization, Qijuan Zang; formal analysis, Yalin Ju; designed the study and funding acquisition, Yingli He; immunofluorescence experiments, Siyi Liu; methodology, Chengbin Zhu, Liangru Liu, and Weicheng Xu; writing—original draft, Yalin Ju, Qijuan Zang, and Siyi Liu; writing—review and editing, Shaobo Wu. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Yingli He.

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The authors declare no conflicts of interest.

Ethics approval

Two groups of human liver tissue specimens were collected from patients of the First Affiliated Hospital of Xi'an Jiaotong University. Three adjacent normal samples and three hepatitis B-related hepatocellular cancer samples were chosen for analysis. The clinical diagnoses were confirmed by pathobiology. All specimens were fixed in formalin and paraffinized for immunofluorescence (IF). Prior to participation in the study, all research participants provided written informed consent. The study protocol complied with the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University (No. XJTU1AF2019LSL-026).

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Zang, Q., Ju, Y., Liu, S. et al. The significance of m6A RNA methylation regulators in diagnosis and subtype classification of HBV-related hepatocellular carcinoma. Human Cell 37, 752–767 (2024). https://doi.org/10.1007/s13577-024-01044-3

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