The incidence of primary liver cancer ranks 6th among all tumors and 4th among cancer-related deaths. In 2018, there were approximately 841,000 new cases of primary liver cancer worldwide, and 782,000 deaths related to liver cancer . Hepatocellular carcinoma (HCC) accounts for about 75–85% of primary liver cancer. Most HCC patients have reached the advanced stage when they are diagnosed, lose surgical opportunities and have a poor prognosis. The 5-year overall survival rate (OSR) is 20% . The main treatments for HCC include surgical resection, liver transplantation, transarterial chemoembolization (TACE) and radiofrequency ablation (RFA), supplemented by systemic chemotherapy and targeted drug therapy . Because early liver cancer no obvious symptoms or atypical clinical manifestations, more than 80% of patients are already in the advanced stage at the time of diagnosis and cannot receive radical treatment .Alpha-fetoprotein (AFP) is the most commonly used biomarker in HCC monitoring and diagnosis, but it has limited specificity and sensitivity in detecting early HCC . The combination of AFP and serum GP73 is better than AFP alone in the early screening of HCC .Serum AFP and AFP-L3 combined with ultrasonography can significantly improve the sensitivity of liver cancer diagnosis (94.3%) .Using serum AKR1B10 alone to diagnose early HCC has a sensitivity of 61% and a specificity of 86%, which is better than AFP alone. The combination of the two AUROC is as high as 94%, which can be vigorously promoted as a marker for early liver cancer diagnosis . However, there are no relevant reports on the immune and inflammation-related markers in serum. At present, the prediction of liver cancer models related to iron death, DNA methylation, m6A methylation, long non-coding RNA, endoplasmic reticulum stress and glycolysis has good predictive effects [31–36].The prognostic model constructed by immune and inflammation-related genes screened in this study has an AUC of 77.9% (Figure2D), which also has a good predictive effect.
In this study, we analyzed the expression of 526 immune and inflammatory response-related genes in HCC tissues, and selected 50 DEGs from the TCGA cohort (Figure 1A). In univariate Cox analysis, 11 DEGs were associated with OS (Figure 1C, D).Prognostic models of 8 immune and inflammatory response-related genes were constructed through LASSO regression analysis and verified in the ICGC cohort. According to the median risk score, patients were divided into high-risk groups and low-risk groups. Independent prognostic analysis showed that risk score is an independent predictor of OS (Figure4).
The prognosis model established in this study includes 8 immune and inflammatory response related genes (CCNF, DNASE1L3, ENTPD2, MFAP2, SLC16A3, SLC7A11, SPP1, STMN1). Except for DNASE3L3, these genes are all up-regulated in HCC tumor tissues and are associated with poor prognosis (Figure3).Cyclin F (CCNF) has been shown to regulate the cell dNTP pool and maintain the stability of the genome by interacting with ribonucleotide reductase family member 2 (RRM2) . Patients with low CCNF expression may have a shorter survival time than those with high expression (P=0.001), and have a higher tendency to relapse (P=0.037) . DNASE1L3 is very important for DNA catabolism and apoptosis. It combines with DNASE1 and plays a key role in the neutrophil extracellular trap and cfDNA degradation, thereby reducing organ damage after inflammation . The up-regulated expression of DNASE1L3 alleviates the accumulation of cytoplasmic DNA under DDR activation conditions, which in turn leads to cell senescence and SASP inhibition, and tumor angiogenesis is impaired. These results indicate that DNASE1L3 is a potential biomarker for predicting the prognosis of HCC , and the positive expression of DNASE1L3 can be used as a key indicator of a good prognosis after liver cancer . E-NTPDases are transmembrane exonucleases on the surface of CD39 superfamily cells, which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and regulating purine signaling . IL-6 transcription down-regulates the expression of ENTPD2 in portal vein fibroblasts without inducing myofibroblast differentiation. Microfibril-associated protein 2 (MFAP2), also known as microfibril-associated glycoprotein 1 (MAGP1), is a component of extracellular elastic microfibrils and interacts with fibrin to affect the function of microfibrils . MFAP2 is significantly up-regulated in HCC, and may promote the development and progression of HCC through its interaction with mutant fibrillin-1. DNMTs inhibitors can down-regulate MFAP2, and MFAP2 may become a potential immunotherapy target for HCC patients .SLC16A3 has been proven to be a downstream factor of TSTA3 immune response-mediated metabolic coupling cell cycle and non-neoplastic hepatitis/cirrhosis tissue replication . The SLC7A11 gene is located on human chromosome 4 and contains 14 exons. It is widely expressed in tissues and cells such as brain, liver, macrophages and retinal pigment cells. Down-regulation of SLC7A11 in HCC cells may increase intracellular ROS levels. In order to inhibit cell proliferation in vitro, the overexpression of SLC7A11 may be related to the advanced pathological stage of liver cancer . Secreted phosphoprotein 1 (SPP1) In tumors, alternative splicing variants are associated with many malignant characteristics in cancer, such as epithelial-mesenchymal plasticity, cancer cell stem cell resistance, chemotherapy resistance and radioresistance, in HCC Among them, SPP1 is an effective prognostic biomarker, which may induce chemotherapy resistance by regulating the autophagy of HCC cells . In HCC, the high expression of STMN1 is positively correlated with higher AFP levels, tumor size, vascular invasion and intrahepatic metastasis, lower 5-year survival rate and early recurrence rate. The MET inhibitor crizotinib can effectively inhibit the crosstalk between cancer cells and stellate cells caused by the HGF/MET pathway triggered by STMN1, and can effectively slow down the tumor growth caused by the high expression of stmn1 .
In order to understand the relationship between risk scores and immune cells and functions, we studied the role of risk scores in the types of immune infiltration. The results showed that the high-risk score was significantly related to C1, while the low-risk score was clearly related to C3 (inflammation) (Figure?), suggesting that C1 promotes the occurrence and development of tumors, and C3 is a good protective factor. It shows that immunity and inflammation play an important role in tumors. In terms of the association between risk score and clinical characteristics, we found that the overall OS of the high-risk group, Grade3-4 and StageIII-IV was significantly shortened (Figure7E).In KEGG enrichment analysis, the main signaling pathways, cell cycle, human T-cell leukemia virus 1 infection, HIF-1 signaling pathway, PPAR signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, cancer metabolism are closely related to tumor occurrence and development the pathways are significantly enriched in high risk (Figure10).
In the high-risk group, the immune scores of aDCs, macrophages, Tfh cells, Treg cells and Th1 cells were higher (Figure7). There are a large number of inflammatory cells infiltrated in tumor tissues, among which macrophages have tumor-promoting and immunosuppressive effects . The immunosuppression of Tfh cells, Treg cells and Th1 cells in the tumor microenvironment leads to poor prognosis of liver cancer .Programmed cell death protein-1 (PD-1) is mainly expressed on the surface of activated T cells, B cells and macrophages. PD-1 inhibits the activation of antigen-specific T cells by binding to its ligands PD-L1 and PD-L2. When PD-L1, which is highly expressed on the surface of tumor cells, binds to PD-1 on the surface of immune cells, it will cause tumor immune escape .CTLA-4 inhibitors induce T cell anergy, thereby inhibiting the anti-tumor immune response . TLR7/8 agonists promote NK-DC crosstalk and enhance the anti-tumor effect of NK cells in hepatocellular carcinoma . sGPC3 attenuates the anti-tumor activity of CAR-T cells in vitro and in vivo, possibly through the combination of membrane-bound GPC3 and CAR-T cells, which leads to immune escape .
Cancer stem cells (CSCs) have been shown to be the cause of HCC  recurrence, metastasis, and resistance to local and systemic treatments. Among them, the HIF-1 signaling pathway, Akt signaling pathway and IL-17 signaling pathway lead to an increase in the proportion of CSCs in liver cancer tissues, leading to immunosuppression . Our research found that MFAP2 is negatively correlated with RNAss and DNAss, and STMN1 is positively correlated with RNAss and DNAss. It shows that MAPF2 may inhibit the differentiation of cancer stem cells, and STMN1 promotes the differentiation of cancer stem cells, but its mechanism of promoting tumor proliferation and invasion is worthy of further study. (Figure8), but they promote tumor proliferation and invasion. The specific mechanism is worthy of further study. Prognostic gene expression also has a certain correlation with stromal score and immune score. CCNF, DNASE1L3, ENTPD2, MFAP2, SLC16A3, SPP1, STMN1 have a strong correlation with Stromal score, indicating that they may be secreted by stromal cells or involved in stromal correlation. DNASE1L3, MFAP2, SLC16A3, and SPP1 are closely related to the immune score, indicating that they are closely related to tumor immunity.This study found that prognostic gene expression also has a certain correlation with Stromal score, immune score and ESTIMATE score. DNASE1L3, MFAP2, SLC16A3 and SPP1 are positively correlated with interstitial score and immune score, indicating that they may be related to interstitial cells and immune cells [58–60].
Using data from the NCI-60 cell line, we found that the increased expression of some prognostic genes is associated with the increased resistance of some FDA-approved chemotherapeutic drugs, such as oxaliplatin, cyclinamide, epirubicin, nelarabine and fluoride. Of course, various prognostic genes are also related to the increase in drug sensitivity of some drugs.
For example, the increased expression of DNASE1L3 and the resistance of liver cancer cells to oxaliplatin, sorafenib combined with oxaliplatin, fluorouracil and calcium folinate in the treatment of portal vein invasion of hepatocellular carcinoma and clinical trials .The expression of SLC7A11 can increase the sensitivity of liver cancer to arsenic trioxide (ATO). Studies have reported that ATO can effectively induce the differentiation of CSCs by down-regulating CSC-related genes, inhibiting the tumorigenic ability of CSCs. The combination therapy of ATO and 5-FU/cisplatin can significantly improve the therapeutic effect of liver cancer cells. ATO and 5-FU/cisplatin synergistically inhibit LIF/JAK1/STAT3 and NF-kB signaling pathways are the potential molecular mechanisms of their differentiation .