HCC is one of the leading causes of cancer-related deaths with highly malignant, recurrent, metastatic, drug-resistant, and late diagnosis . Thus, the identification of effective biomarkers for HCC-specific prognosis is urgently needed to improve the management for patients. Recently, global alterations in metabolic pathways were identified in HCC , providing some new diagnostic and therapeutic opportunities . Taking into account the importance of the alteration of metabolic processes in HCC progression and the highly central position of the mitochondrion in human metabolism, it is essential to identify mitochondrial-related biomarkers for the prognosis of HCC patients, which may also help us to clarify underlying metabolism alterations and identify potential therapeutic drugs, so as to bring new insights into the improvement of the prognosis of HCC patients.
In the present study, a 10 Mito-RGs based prognostic classifier for HCC was constructed and validated to optimize the predictive capacity of prognosis for HCC patients for the first time. The classifier did well in predicting the prognosis for HCC patients in training, validation, and total cohorts, indicating the repeatability and practicability of the classifier for the prognostic prediction for HCC overall survival. Besides, the prediction efficacy of the classifier was superior to histologic grade and tumor stage (TNM stage), which may be two major risk factors for tumor prognosis as reported by previous studies [15, 16].
All these 10 Mito-RGs of the classifier, ACOT7, ADPRHL2, ATAD3A, BSG, FAM72A, PDK3, PDSS1, RAD51C, TOMM34, and TRMU, were risk-associated, and higher expressed in high-risk group. Besides, among them, ACOT7, ADPRHL2, ATAD3A, BSG, FAM72A, PDSS1, RAD51C, and TOMM34 were overexpressed in HCC compared with normal liver tissues, indicating potential roles of these genes in the initiation and development of HCC. ACOT7 is an isoform of the ACOT family, which responsible for cleaving fatty acyl-CoAs to free fatty acids that involved in responses to endoplasmic reticulum stress in lung cancer . Besides, the high expression of ACOT7 indicates unfavorable outcome in acute myeloid leukemia patients . ADPRHL2, also known as ADP-ribosylhydrolase 3 (ARH3), is the main hydrolase for catalyzing the hydrolysis of ADP-ribosylated serine. The deficiency of ADPRHL2 will enhance the tumor-suppression effect of ARH1 , indicating the potential carcinogenic role of ADPRHL2. ATPase family AAA domain-containing protein 3 (ATAD3A) is a mitochondrial membrane-bound ATPase, involving in various cellular processes . Besides, the high expression of ATAD3A is correlated with the poor prognosis of HCC . BSG, also termed as CD147, is a member of the immunoglobulin superfamily that is overexpressed and positively correlated with HCC malignant potential and poor prognosis . FAM72A, also known as p17, is a novel neuronal protein that also exerts tumorigenic effects in multiple tissues  by modulating cell survival and promoting the cell cycle . RAD51C is one of the paralogs of RAD51 and is essential for homologous recombination . High expression of RAD51C predicts poor prognosis and is correlated with resistance to chemoradiotherapy in lung cancer . TOMM34 is a kind of protein located in the outer membrane of mitochondria (TOMM), which overexpressed in breast cancer and served as a biomarker of the progression and poor prognosis of breast cancer . However, the role of these genes in HCC mostly unclear. Considering the strong relevance to the prognosis of HCC, these genes highly worthy of further investigation in HCC.
Metabolic alterations are a well-founded hallmark of HCC . The wide range of metabolic alterations is highly associated with the heterogeneity of HCC, providing challenges for the clinical management of HCC patients . In the present study, we found that the metabolic pathways about fatty acid metabolism, amino acid metabolism, and bile acid biosynthesis were downregulated in high-risk HCC patients, indicating the potential role of these metabolic processes in the progression of HCC.
The dysregulation of de novo lipogenesis is a well-known characteristic of cancer metabolism, which ensures the energy supply for the proliferating tumor cells . The liver has a central role in the processes of lipids synthesis, storage, and degradation. The imbalance of fatty acid metabolism induced by upregulation of de novo lipogenesis and downregulation of lipid degradation in the liver will lead to the accumulation of lipids, and can rapidly develop the nonalcoholic fatty liver disease (NAFLD), which is a risk factor for HCC . Studies showed that the upregulation of the lipogenic metabolism genes, including fatty acids biosynthesis enzymes (ACAC, ACLY, FASN, and SCD1), enzymes for cholesterol biosynthesis (HMGCR, MVK, SREBP2, and SQS), as well as their upstream transcriptional factors (chREBP, LXR-β, and SREBP1) was observed in HCC when compared with adjacent normal liver tissues . Besides, the higher levels of lipogenic enzymes were associated with poorer prognosis . In the present study, we also found the metabolic process of fatty acid degradation was downregulated in high-risk group of HCC, and its high level correlated with a better prognosis of HCC patients. These data indicated that increased lipogenesis and downregulated lipid degradation will promote the development and progression of HCC, which may be potential therapeutic targets for HCC.
Amino acids (AAs) perform important metabolic functions, which can be divided into essential AAs, including histidine, isoleucine, lysine, leucine, methionine, phenylalanine, tryptophan, threonine, and valine, semi-essential AAs, including arginine, cysteine, glutamine, glycine, tyrosine, and proline, non-essential AAs, including alanine, aspartate, asparagine, glutamate, and serine . They are not only components for protein synthesis but also intermediate metabolites for multiple biosynthetic pathways. Besides, increasing studies have shown that AAs also functioned as metabolites and metabolic regulators in supporting cancer cell growth .
The alterations of AAs metabolism were characterized in HCC compared to other liver diseases. Research has shown that the alteration of urine metabolites in HCC patients was mainly involved in arginine and proline metabolism, glutathione metabolism, phenylalanine metabolism, and tyrosine metabolism, which may be considered as potential biomarkers . Metabolites of AAs such as tyrosine (OR = 4.11), glutamate (OR = 3.93), glutamine (OR = 0.15), kynurenine (OR = 3.32), lysine (OR = 0.35), and leucine (OR = 0.19), isoleucine (OR = 0.51), valine (OR = 0.56), AA groups and metabolite ratios such as branched-chain (OR = 0.26), glucogenic (OR = 0.25) and aromatic AA (OR = 3.29), and the Fischer's ratio (branched-chain to aromatic AAs) (OR = 0.10), glutamate/glutamine (OR = 5.49) and kynurenine/tryptophan (OR = 2.29) ratios were found significantly associated with HCC risk . In the present study, the pathways of Glycine, serine and threonine metabolism, Histidine metabolism, Tyrosine metabolism, Tryptophan metabolism, and Valine, leucine and isoleucine degradation were found downregulated in high risk of HCC, and low level of these metabolic processes was correlated with poor prognosis.
Serine and glycine are linked in the biosynthetic process that serine is formed prior the glycine. They together provide the essential precursors for glucose, lipids, proteins, and nucleic acids synthesis through one-carbon metabolism that are crucial to cancer cell growth and support tumor homeostasis . The upregulation of serine/glycine metabolism promotes cell proliferation and correlates with poor prognosis in various tumors [36, 37]. Metformin has recently acted as a promising agent for cancer therapy. A recent study revealed that metformin exerts anti-tumor effects through the alteration of one-carbon metabolism .
Tyrosine metabolism has been found altered in esophageal squamous cell carcinoma (ESCC) patients and the metabolites involved in can be utilized as diagnostic biomarkers . Besides, studies showed that 4-hydroxyphenylpyruvate dioxygenase (HPD) played important roles in tumorigenesis through promoting tyrosine metabolism . Researches showed that activation of tyrosine metabolism in CD13+ cancer stem cells triggers the relapse of HCC .
Tryptophan metabolism is involved in the regulation of immune function, neuronal function, and intestinal homeostasis. Studies have shown that tryptophan metabolism played important roles in promoting tumor progression by contributing to tumor immune escape and increasing the malignancy of cancer cells . Indoleamine 2,3 dioxygenase 1 (IDO1) is the most important enzymes that initiate the catabolism of tryptophan to kynurenine, which is expressed in 58% of human tumors , and its expression is correlated with poor prognosis in various cancers including melanoma, colorectal cancer, ovarian cancer, brain tumors, and acute myelogenous leukemia .
Leucine, isoleucine, and valine are essential AAs of branched-chain amino acids (BCAAs), and they have recently served as predictive factors for the risk of cancers . BCAAs metabolism is altered in various tumors, including melanoma, breast cancer, and nasopharyngeal carcinoma . BCAAs are thought to play important roles in cancer cells through activating of the mammalian target of rapamycin complex 1 (mTORC1), which promoting protein synthesis, cell growth, and proliferation . Besides, they can also provide the essential precursors for nucleic acids synthesis and tricarboxylic acids cycle (TCA) that contribute to energy production for cancer cell growth . Additionally, the role of branched-chain aminotransferase 1 (BCAT1), the key enzyme that mediates BCAAs catabolism, played essential roles in cancer progression. In breast cancer , lung cancer , ovarian cancer , HCC , and chronic myeloid leukemia , BCAT1 levels are increased compared to normal tissues and promote cell proliferation through activating mTORC1, inhibiting autophagy, suppression isocitrate dehydrogenase 1(IDH1), IDH2, and aldo-keto reductase family 1 member C1 (AKR1C1). Besides, a recent meta-analysis revealed that BCAAs supplementation improved liver functional, reduced 3-year mortality for HCC patients .
Primary bile acids are synthesized from cholesterol in the liver by the classic pathway and the alternative pathway. The classic pathway accounts for about 90% of total bile acid production in the liver (produces cholic acid (CA) and chenodeoxycholic acid (CDCA)), mainly catalyzed by cholesterol 7α-hydroxylase (CYP7A1) . While, the alternative pathway is catalyzed by CYP27A1 and CYP7B1, produces chenodeoxycholic acid (CDCA) . Early in the 1970s, a study has shown that plasma bile acid concentrations are elevated in HCC patients compared to healthy individuals , indicating that bile acid homeostasis was disturbed during HCC. Studies have shown that the knockout of whole body Farnesoid X receptor (FXR), which is an endogenous ligand for bile acids and transcriptional downregulates the expression of the enzymes of bile acid synthesis such as CYP7A1, CYP27A1, and CYP8B1 , would develop liver tumors in mice . Increasing evidence revealed that bile acids could directly disrupt the plasma membrane and activate the MAPK-NF-κB pathway, resulting in the increase of TNF-α, IL-1β and IL-6. These cytokines could activate the JAK-STAT3 pathway, and PI3K-MDM2 pathway, which increases survival of DNA damaged cells and leading to the development of HCC. Besides, membrane injury by bile acids can also lead to the increase of reactive oxygen species (ROS) in the hepatocytes by activating cytosolic phospholipase A2 (PLA2), which can directly activate NF-κB and can also induce direct DNA damage in cells which might lead to HCC . In the present study, we found that a high level of primary bile acid synthesis was associated with a better prognosis of HCC, which seems inconsistent with previous studies. The result may be due to the inhibitory role in proliferation and regeneration , and the induction of cell death by membrane disruptions  by the high concentration of bile acids, which may repress the progression of HCC. According to this, multiple synthetic bile acid derivatives were designed and found useful for cancer therapy .
Inevitably, the present study has some limitations. Firstly, it was a retrospective study based on the publicly online database. Secondly, the predictive role of our classifier was not confirmed by the cohort GSE76427 from GEO database (Gene Expression Omnibus) (Additional files 2: Supplementary Fig. 4). We further found that there existed a significant censoring proportion in the survival data of the GEO cohort. In which 14.8% patients were censored within 1 month, 35.7% within 1 year, and 47.8% within 2 years. Thus, we believed that our classifier could be used as a reliable prognostic predictor for HCC survival, since it was come from a large, high-quality database. Therefore, large-scale, multi-center studies are needed to verify our results before the Mito-RGs based classifier can be applied in the clinic.