Hepatocellular carcinoma is one of the common primary tumors, causing more than 700,000 deaths each year. Tobacco, heredity, epigenetic factors will affect its development. Although many detection and treatment methods of computer tomography (CT) and emerging carbon ion radiation have made good progress (Malouff et al., 2020). HCC patients are prone to recurrence and metastasis of hemangioma after surgery or ablation, causing the poor prognosis (Korean Liver Cancer Association and National Cancer Center, 2019; Brown et al., 2019; Muto et al., 2014). Currently, the study’s diagnostic markers are limited and only for some populations (Rawat et al., 2018). Therefore, it is of great significance to study specific diagnostic biomarkers to predict the survival and diagnosis of HCC.

Epigenetic regulation does not change DNA sequence but changes the transcription mechanism and affects the tumor’s progress (Sharma et al., 2020; Dzobo et al., 2020; Baylin and Jones, 2011). Among them, DNA methylation (5-methylcytosine DNAm, m5C) and RNA methylation (6-methyladenine RNAm, m6A) are two crucial nucleic acid modifications, which are epigenetic important regulation mechanism (Liu and He, 2020). DNAm modification affects gene transcription (alternative splicing or translation), leading to abnormal gene expression and causing diseases. DNA methylation sites were served as prognostic markers in colorectal cancer and kidney cancer with high sensitivities, e.g., cg10673833 and cg04448376 (Luo et al., 2020; Wang and Zhang, 2020; Ibrahim et al., 2011; Ashokan et al., 2021). Compared with a single omics biomaker to explore HCC prognosis, the strategy combining DNA methylation data and transcriptomics’ data is believed to be more promising.

The APLN gene locates on chromosome Xq25-q26.1 and can encode 77 amino acid pro-peptide Apelin (Xu et al., 2017). APLN may be a potential marker in ovarian cancer, colorectal cancer, kidney cancer and other cancers (Chen et al., 2021; Ran et al., 2021; Bai et al., 2020; Zuurbier et al., 2017). Not only can APLN gene activate the pathway of APLN/APLNR to promote the HCC development, but also it activates different signal pathways or plays anti-apoptotic and anti-inflammatory functions by promoting tumor angiogenesis (Cabiati et al., 2021; Wang et al., 2020b). Many factors can affect the development of HCC. Immature and unstable tumor vasculature is associated with abnormal tumor microenvironment, which is liable to pose drug resistance during the anti-cancer treatment. The new peptide apelin and its receptor can enhance immune efficacy by inducing the morphological and functional maturation of tumor blood vessels (Kidoya et al., 2012). APLN/APLNR will be up-regulated during tumor angiogenesis. And APLN stimulates the proliferation, migration and invasion of colon adenocarcinomas (Picault et al., 2014; Podgorska et al., 2018). In addition, increased expression of APLN has been observed in muscle-invasive bladder cancer and is associated with poor clinical outcomes (Yang et al., 2019). Studies found that epigenetic modification and Expression of APLN can affect the progression of lung cancer (Miller et al., 2018). The degree of CpG island DNAm in APLN is related to the infiltration of pneumonia cells. And the DNA methylation sites in important regions can regulate gene function (Mishra et al., 2015). APLN can inhibit tumor progression by regulating Tregs immune cells’ expression and VCAM-1 (Amoozgar et al., 2019). We studied the expression changes of APLN in the development of HCC and its ability to predict prognosis. The methylation sites signatures in epigenetics are used to predict the viability of HCC, which is closely related to APLN.

Our team determined the molecular interaction mechanisms and biomarkers of certain diseases through bioinformatics methods (Tian et al., 2015). This study is based on the transcriptome, genetic data of the TCGA dataset and the UCSC dataset to study the prognosis of HCC. First, we used GEPIA and TIMER to screen out suitable genes. Then we screened out APLN gene through PubMed. CIBERSORT’s deconvolution can estimate the abundance of 22 kinds of immune cells and differential immune cells. Immune cells and checkpoints are used as clinical traits for WGCNA. And the univariate Cox, Lasso Cox were utilized to construct the risk score. The Nomogram was used to identify the prognostic ability of DNAm signature. The flow chart of this research is shown in the Fig. 1.

Conventional treatment for HCC is highly invasive. And early prediction and personalized therapy of targeted immune checkpoints have research significance (Yin et al., 2019; Wang et al., 2021). APLN and DNAm are abnormally expressed in a variety of cancers. But the role of APLN in the immune microenvironment development of HCC is still unknown. APLN is up-regulated in HCC and HCC38/KMUH cells and may be used as a therapeutic target (Lin et al., 2012; Chen et al., 2019). APLN is a carcinogen that is highly expressed and its expression trend is increasing with HCC development. APLN, as a transcriptional target of the Wnt/β-catenin pathway, promotes HCC by activating PI3K/Akt signaling. The pharmacological targeting of APLN strongly inhibits the proliferation of HCC cells and tumor growth, which means that APLN may be a potential drug target for HCC. The gene has significant differences in the expression and clinical characteristics between the tumor and normal tissues. It has the potential prognostic value. The Cox regression model predicts the risk prediction characteristics significantly related to OS. Finally, the prognostic value of the risk prediction was verified by the nomogram.

The study determined that the proportion of total T cells in HCC cells is greater than in healthy tissues. And T cell subsets mainly include T cell CD4 memory resting, follicular helper T cells, etc., which impact the prognosis of HCC (Jia et al., 2015a; Rohr-Udilova et al., 2018; Garnelo et al., 2017). APLN affects three types of immune cells, including T cell CD4 memory resting, follicular helper T cells, and Tregs, highlighting the poor clinical correlation between T cell-mediated immunity and tumors (Li et al., 2021). Both PD-1 and CTLA-4 are related to some subsets of T cells, and mainly affect the development of tumors through PD-1/PD-L1 acting on T cells (Kim and Chen, 2016). PD-1 and PD-L1 interaction can resulting in inactivation of the immune response (Poureau and Metges, 2021). PD-1 is the main checkpoint in TME. Follicular helper T cells can inhibit the expression of Treg cells by affecting the expression of PD-1. And CTLA-4 can also enhance the immunosuppressive activity of Treg (a key mediator of CTLA-4) by regulating follicular helper T cells (Jia et al., 2015b; Hervouet, 1970; Yang et al., 2014; Topalian et al., 2016). The expression of APLN and specific immune cells play an essential role in the tumor progression of HCC (Wang et al., 2020a). APLN can regulate or induce the morphological and functional maturation of tumor blood vessels. And the recruitment of dendritic immune cells helps to enhance the effect of immunotherapy (Kidoya et al., 2012). Closely related to the HCC prognosis, the immune microenvironment is complex and usually heterogeneous with several genomic alterations, therefore making meaningful the integrated investigation of immune microenvironment with the DNA methylation sites of APLN in studies of HCC prognosis.

Not only does tumor-related macrophages promote thrombosis and endocytosis, angiogenesis, invasion and metastasis, but also it inhibits anti-tumor immune responses (Lin et al., 2019). Tumor-associated macrophages are mainly polarized into Macrophages M1 and Macrophages M2. The former plays an anti-tumor effect, while the latter has a tumor-promoting effect. Macrophages M2 mainly induces anti-inflammatory responses by producing IL-10 and (TGF)-β, meanwhile participating in the polarization of Th2 responses to promote tumor growth (Wildes et al., 2020; Biswas and Mantovani, 2010; Wolf-Dennen et al., 2020). Taking a higher percentage in HBV, non-polarized Macrophages M0 also affect the HCC prognosis (Ding et al., 2021; Huo et al., 2020). APLNs induce tumor vascular maturation and promote the infiltration of natural killer T cells into the central region of the tumor. This process enhances the efficacy of cancer dendritic cell-based immunotherapies (Mastrella et al., 2019). APLN inhibits and reduces neutrophil recruitment, thereby reducing the inflammatory and fibrotic response of pancreatitis. It also inhibits macrophage infiltration and monocytes, thereby enhancing the stable phenotype of atherosclerotic plaques (Ioannidis et al., 2010).

The signatures that DNA methylation sites analyzed by LASSO COX prevent data from overfitting. Studies found that the combination of selected central genes and DNAm conditions has an advantage in predicting the survival of HNSCC (Mou et al., 2020; Cao et al., 2020). The human m6A near the mRNA stop codon and the 3’ untranslated region will affect the transcription and function of genes through the promoter DNAm (Ondo et al., 2021). Usually, DNAm inhibits gene expression. APLN and the probe cg05688478 can affect gene expression in human pancreatic islets (Hall et al., 2014). The interaction of genome (gene expression) and epigenome (DNAm level) to control Apelin endothelium is one of the most critical ways for non-triple-negative breast cancer (Wu et al., 2020). DNA methylation silences the APLN expression, leading to increased insulin secretion in women (Hall et al., 2014). In APLN, rs3761581 G and rs2235312 T are relative to the degree of CpG island. Low levels of apelin-13 will cause greater methylation of CpG islands in the 5’UTR, which contributes to the pathophysiology of HA lung water. Abnormal methylation may prevent apelin from binding to hypoxia response elements, thereby inhibiting APLN transcription (Mishra et al., 2015). In downstream of DNA damage, the increased methylation of the APLN promoter may lead to reductions in protective signaling of the apeliner energy system. Then it lead to the pulmonary edema observed after exposure to oxidative air pollution (Miller et al., 2018).

The interaction between the genome and the epigenome will also change the evolution and control the development of different cancers. Epigenetic controlled immunotherapy has potential effects such as restoring anti-tumor signals (Gomez et al., 2020). Many sites in the DNAm region and epigenetic changes play an essential role in the early stage of liver cancer (Seal et al., 2020). The DNAm mechanism would regulate the immune cell response of cancer. Different DNAm patterns will also affect the characteristics of immune infiltration (Wu et al., 2020; Du et al., 2021). Epigenetics and gene expression influence each other (Mishra et al., 2015; Wang et al., 2020c). Studies have found that the CpG island of APLN has a more significant impact on lung tissue. The combined study of APLN and epigenetic DNA methylation sites are significantly related to survival. Research using epigenetics, gene transcriptome, and immune data, predicting prognostic markers of HCC has great potential.

The study still has limitations. The samples we studied are entirely retrospective. The data bias may affect survival and DNAm signatures. Although we determined the selected DNA methylation sites and APLN as predictive features, more experimental studies are still needed.

In summary, our research combines transcription, epigenetics, and immune data to predict HCC prognosis. The novel biomarkers immune-related APLN may be reliable prognostic markers for HCC.