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Construction and validation of prognostic signature for hepatocellular carcinoma basing on hepatitis B virus related specific genes



Hepatocellular carcinoma (HCC) is a frequent primary liver cancer, and it is one of the leading cause of cancer-related deaths. Hepatitis B virus (HBV) infection is a crucial risk factor for HCC. Thus, this study aimed to explore the prognostic role of HBV-positive HCC related specific genes in HCC.


The HCC related data were downloaded from three databases, including The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO). Univariate Cox regression analysis and LASSO Cox regression analysis were conducted to build the Risk score. Multivariate Cox regression analysis and survival analysis determined the independent prognostic indicators.


After cross analysis of differentially expressed genes (DEGs), we have identified 106 overlapped DEGs, which were probably HBV-positive HCC related specific genes. These 106 DEGs were significantly enriched in 213 GO terms and 8 KEGG pathways. Among that, 11 optimal genes were selected to build a Risk score, and Risk score was an independent prognostic factor for HCC. High risk HCC patients had worse OS. Moreover, five kinds of immune cells were differentially infiltrated between high and low risk HCC patients.


The prognostic signature, based on HMMR, MCM6, TPX2, KIF20A, CCL20, RGS2, NUSAP1, FABP5, FZD6, PBK, and STK39, is conducive to distinguish different prognosis of HCC patients.


Hepatocellular carcinoma (HCC) is a frequent primary liver cancer, and it has ranked the second leading cause of various cancers’ mortality up to 2020 [1]. The incidence of HCC has been reported to increase during the past decades, and over 90,000 new cases of HCC are estimated in 2020 [2]. Many risk factors for developing HCC have been widely investigated, such as non-alcoholic steatohepatitis, chronic hepatitis B or C virus infections, and progressive fibrotic liver diseases [3,4,5]. Only a small part of HCC patients (about 20%) could be diagnosed at an early stage, and these patients are more probably eligible for surgical therapies or radiofrequency ablation [6]. As for those undetectable HCC patients, oral tyrosine-kinase inhibitor (TKI) sorafenib has been the first-line treatments with survival benefit and enough safety [7, 8]. However, due to high metastasis and recurrence rate, the long term prognosis of HCC patients is still poor, and the 3-year and 5-year overall survival (OS) rate is less than 20% [9, 10]. Accordingly, increasing diagnostic or prognostic biomarkers/ signatures are expected to improve the outcome of HCC patients directly or indirectly, such as hypoxia-related prognostic signature [11], immune-related signature [12], and so on.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are dominating risks causing HCC, among which HBV is a heavier healthy burden in China [13,14,15]. HBV, as a small hepatotropic DNA virus, could result in acute or chronic liver diseases, thereby leading to hepatic damage, fibrosis and liver cancer [16, 17]. Before the transformation from HBV infection to HCC, there is a long-time interaction between HBV and host hepatocytes, comprising HBV DNA integration, aberrant regulatory protein expression, and epigenetic dysregulation [18]. Moreover, in most adult patients, HBV infection could lead to a rapid immune response and acute self-limited infection [19]. Currently, increasing HBV based biomarkers or signatures have suggested their favorable potential regarding the prognosis or diagnosis of HCC patients. PYCR2 (pyrroline-5-carboxylate reductase 2) and ADH1A (alcohol dehydrogenase 1 A (class I), alpha polypeptide) are recently identified as prognostic biomarkers in HBV-related HCC, involving metabolic reprogramming [20]. Moreover, Yan et al. have built an OS predictive signature based on 4 genes in HCC [21]. A two-m6A-regulator based prognostic signature has been reported in HBV-related HCC [22]. Whereas, as far as we know, few prognostic signatures based on HBV related specific genes in HCC have been reported.

In this study, we mainly aimed to explore the prognostic value of HBV-positive related genes in HCC patients. Integrating HCC data from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO) databases, multiple bioinformatic analyses were conducted in order to construct a reliable prognostic signature for HCC. Our study is expected to be helpful to predict or partly improve the prognosis of HCC patients.


Research objects

Mutation Annotation Format (MAF) files of 365 HCC patients were downloaded from The Cancer Genome Atlas (TCGA) database ( Then we also downloaded the mRNA expression profile and the corresponding clinical information of 371 HCC patients from TCGA database, of which 365 patients had complete survival information (detailed clinical information was shown in Table 1). Moreover, other 237 HCC patients’ clinical information and mRNA data were obtained from Liver Cancer-RIKEN JP (LIRI-JP) dataset in International Cancer Genome Consortium (ICGC) database (

Table 1 Clinicopathological characteristics of HCC sampless from TCGA database

Additionally, another two datasets were downloaded from Gene Expression Omnibus (GEO) database ( GSE83148 comprised 6 normal liver tissue samples and 122 hepatitis B virus (HBV) infected liver tissue samples, totally 128 samples. GSE121248 contained 107 samples, including 37 HBV-positive HCC patients’ adjacent samples and 70 HBV-positive HCC patients’ tumor samples. The data in these two datasets were both detected on Affymetrix Human Genome U133 Plus 2.0 Array platform.

LASSO Cox regression analysis

Based on gene expression, the HCC samples were subjected to univariate Cox regression analysis, after which the genes significantly related to the prognosis of HCC patients were screened with threshold P < 0.01. The optimal HCC prognostic related genes were further selected via LASSO Cox regression analysis using glmnet package of R [23]. Based on the optimal genes, all samples’ Risk score can be calculated via the following formula:

$$\ {\text{Risk}}\;{\text{Score}} = \sum\limits_{{{\text{i}} = 1}}^{{\text{n}}} {{\text{Coef}}_{{\text{i}}} } * {\text{X}}_{{\text{i}}}$$

Coefi was the risk coefficient calculated via LASSO Cox regression analysis, and Xi referred to mRNA expression here.

Then, according to the median of Risk score, all HCC samples were divided into high and low risk groups.

Differential expression analysis

We utilized limma package [24] of R (version 3.5.2) to conduct differentially expressed gene (DEG) analysis. Significant DEGs were screened basing on |log2FC| >1 and FDR ≤ 0.05.

Enrichment analysis

The functional enrichment analysis was then performed on these significant DEGs using “clusterProfiler” [25] of R, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. P value < 0.05 (adjusted by Benjamini and Hochberg (BH) method) was adopted to screen significantly enriched GO terms and KEGG pathways.

Survival analysis

The OS rates of various groups were estimated according to Kaplan-Meier method, utilizing survival and survminer packages of R. The significance of difference was determined by log rank test.

Immune cell infiltration analysis

The relative proportions of various immune cells in every sample were calculated using software CIBERSORT [26]. Basing on gene expression matrix, relative proportions of infiltrating immune cells could be characterized according to the deconvolution algorithm. For each sample, the CIBERSORT output estimated proportions sum up to one.

Nomogram building

Nomogram is an important tool to predict the prognosis of cancer patients. Thus, we utilized all independent prognostic factors obtained from multivariate Cox regression analysis to construct Nomogram, predicting 1, 3 and 5-years OS of HCC patients (using rms (Regression Modeling Strategies) package of R ( The calibration curve was drawn to test the prognostic performance of Nomogram.

Drug target predictions

Genomics of Drug Sensitivity in Cancer (GDSC) database ( has been the largest public database including tumor cell drug sensitivity and tumor treatment genome data. Herein, this database was used to predict the corresponding medication information of genes, in order to explore the correlation between gene and drug sensitivity (ANOVA analysis, P value < 0.05).

Statistical analyses

All independent prognostic indicators for HCC patients were determined by multivariate Cox regression analysis. Immune cell infiltration difference was tested by Wilcoxon signed rank sum test, and p < 0.05 was considered significant. All statistical analyses were conducted in R software v3.5.2.


Identification of HBV-positive HCC related genes

Firstly, basing on the data in GSE83148, we have conducted a differential expression analysis on normal liver tissue and HBV-positive liver tissue samples. Compared with normal liver samples, a total of 614 DEGs were identified in HBV-positive liver samples, comprising 561 upregulated genes and 53 downregulated genes (Fig. 1 A). Additionally, in GSE121248 dataset, compared with HBV-positive adjacent samples, there were 680 DEGs in HBV-positive HCC samples, including 227 upregulated genes and 453 downregulated genes (Fig. 1B).

Fig. 1
figure 1

Identification of HBV-positive HCC related genes. A, B The identified DEGs in GSE83148 dataset and GSE121248 dataset, respectively. C The top 20 significantly enriched GO terms. X-axis: the number of enriched genes; Y-axis: names of GO terms. D Eight significantly enriched KEGG pathways. X-axis: the number of enriched genes; Y-axis: names of pathways

We found that there were 106 overlapped DEGs between these two datasets (Additional file 1: Table S1), which were probably specific genes related to the development from HBV to HCC. In GSE83148, 94 overlapped genes were upregulated, and 12 genes were downregulated in HBV-positive samples. In GSE121248, there were 46 upregulated overlapping genes and 60 downregulated genes in HBV-positive HCC samples. Then the 106 DEGs were significantly enriched in 213 GO terms (top 20 terms, Fig. 1C) and 8 KEGG pathways (Fig. 1D). All detailed functional enrichment results were summarized in Additional file 2: Table S2.

HCC patients with distinct prognosis could be divided based on HBV-positive HCC related genes

Subsequently, basing on the expressions of the 106 HBV-positive HCC related genes, the 365 HCC samples in TCGA database were subjected to cluster analysis. The results of sum of the squared errors (SSE) indicated that the optimal clusters should be k = 4 (Fig. 2 A), then all samples were clustered into 4 categories (Fig. 2B). After conducting Kaplan-Meier (KM) survival analysis, we found significantly differential OS among 4 clusters’ HCC patients. HCC patients in Cluster1 and Cluster2 had worse prognosis, while patients in Cluster3 and Cluster4 had better prognosis (Fig. 2C). After checking the clinical information of these 365 HCC patients, we found that 142 HCC patients were HBV-positive, and the rest 223 patients had unclear HBV information. Regarding the 142 HBV-positive patients, among which, 65 (45.8%) and 41 (28.9%) patients were clustered in poor prognostic Cluster1 and Cluster2, respectively (Fig. 2D). Whereas, only 19 (13.4%) and 17 (11.9%) patients were clustered into good prognostic Cluster3 and Cluster4, respectively (Fig. 2D).

Fig. 2
figure 2

The cluster analysis results of the HCC samples. A Elbow diagram indicated that the optimal number of clusters was k = 4. B The cluster dendrogram of HCC samples. Different colors represent different clusters. C Kaplan Meier survival curve. The P value was calculated based on log-rank test. D The distribution of HBV-positive HCC patients in various clusters

The risk score based on 11 genes could reliably predict the prognosis of HCC patients

All HCC samples in TCGA database was then subjected to an univariate Cox regression analysis taking expression values of 106 HBV-positive HCC related genes as continuous variable, and the Hazard ratio (HR) of each gene was calculated. HR < 1 represented the beneficial role of gene in patient prognosis, while HR > 1 meant higher risk for patient poor prognosis. Then we obtained a total of 42 significant genes (P value < 0.01), all of which were risk genes with HR > 1 (Fig. 3 A). LASSO Cox regression analysis was subsequently performed on these 42 selected genes. According to the lowest lambda value, the corresponding optimal number of gene was 11 (Fig. 3B). The optimal genes included HMMR (hyaluronan mediated motility receptor), MCM6 (minichromosome maintenance complex component 6), TPX2 (TPX2 microtubule nucleation factor), KIF20A (kinesin family member 20 A), CCL20 (C-C motif chemokine ligand 20), RGS2 (regulator of G protein signaling 2), NUSAP1 (nucleolar and spindle associated protein 1), FABP5 (fatty acid binding protein 5), FZD6 (frizzled class receptor 6), PBK (PDZ binding kinase), and STK39 (serine/threonine kinase 39).

Fig. 3
figure 3

The construction of predictive Risk score for HCC. A HCC prognostic related genes. HR: Hazard ratio, 95% CI 95% confidence interval. B The optimal gene number was 11, corresponding to the lowest lambda. X-axis: log(lambda); Y-axis: partial likelihood deviance. C, D Kaplan Meier survival curve of HCC samples in TCGA and ICGC databases, respectively. P value was based on log-rank test. E Multivariate Cox regression analysis results. HR > 1 means higher death risk, while HR < 1 is contrary

Then gene expression was weighted with regression coefficient of LASSO Cox regression analysis to establish a predictive prognostic Risk score model, Risk score = (HMMR*0.142113311)+(MCM6*0.084558199)+(TPX2*0.285390199)+(KIF20A*0.083398740) )+(CCL20*0.029641894)+(RGS2*0.031218559)+(NUSAP1*-0.449566369)+(FABP5*0.002614688)+(FZD6*0.017389305)+(PBK*0.094068871)+(STK39*0.032363425). Thus, the Risk score could be calculated for each sample. All HCC samples, in TCGA database (training set) and ICGC database (validation set), were divided into high and low risk groups, basing on the median of Risk score. We found that in both data sets, HCC patients with high Risk score had poorer OS compared with low Risk score patients (Fig. 3C, D). Moreover, a multivariate Cox regression analysis was then conducted on age, gender, Stage, Grade, Vascular tumour invasion and Risk score in order to find independent prognostic indicators for HCC patients (Fig. 3E). Our results showed that Risk score and Stage were significantly related to OS of HCC patients. Those HCC patients with higher Risk score had poorer OS compared with lower Risk score patients (HR = 3.68, 95%CI 2.38–5.7, P < 0.001). Collectively, the Risk score built based on HMMR, MCM6, TPX2, KIF20A, CCL20, RGS2, NUSAP1, FABP5, FZD6, PBK and STK39, could well predict the prognosis of HCC patients.

Nomogram had good prognostic prediction performance

Nomogram was then built based on the two independent prognostic factors, comprising Stage and Risk score (Fig. 4A). For each HCC patient, three upward lines would determine the Points got from the Nomogram, the sum of the points was located on the Total Points axis. A line downward from Total Points axis finally determined the 1, 3 and 5-years OS of HCC patients. The 1 and 3-years calibration curves were well matched the ideal curve (the line passing through origin with a slope of 1), which implied that the Nomogram had a relatively good prognostic predictive effect (Fig. 4B and D).

Fig. 4
figure 4

Nomogram could predict the OS of HCC patients. A Nomogram could predict 1-year, 3-year, and 5-year OS of HCC patients. BD Nomogram calibration curves of 1-year, 3-year, and 5-year, respectively. X-axis: predicted survival probability; Y-axis: actual survival probability

The differential immune cell infiltration and differential mutated genes between high and low risk HCC patients

Combining LM22 feature matrix with CIBERSORT method, various immune cells’ infiltration was estimated in high and low risk HCC patients. The detailed immune cells’ infiltration of 365 HCC samples in TCGA database has been summarized in Fig. 5 A, which implied that tumor immune cell infiltrating heterogeneity of different individuals. Between high and low Risk HCC patients, totally 5 types of immune cells, including Macrophages M0, Macrophages M2, Monocytes, T cells CD4 memory resting, and T cells regulatory Tregs, were significantly differentially infiltrated (Fig. 5B). We found that in high risk HCC patients, TP53 (tumor protein p53) showed the highest mutation rate (42%) (Fig. 5 C), while in low risk patients, CTNNB1’s (catenin beta 1) mutation rate 25% was highest (Fig. 5D). Meanwhile, the ratio of HBV-positive HCC patients (46.4%) in high Risk score patients was higher than that in low Risk score patients (31.3%). We have searched the medication information targeting TP53 mutation in GDSC database, which indicated that Uprosertib and BMS-536,924 had high sensitive to TP53 mutated HCC patients (Fig. 5E).

Fig. 5
figure 5

Immune cell infiltration difference between high and low risk HCC patients. A Immune cells’ infiltration of 365 HCC samples in TCGA database. B 5 types of significantly differentially infiltrated immune cells between high and low risk HCC patients. C, D The top 20 genes with highest mutation rates in high and low Risk score HCC patients, respectively. E Drug sensitivity results. X-axis: IC50 score


Despite great efforts have been devoted to improve the outcome of HCC patients, little effect has been brought to prolong the OS of HCC patients [27]. In this study, we have integrated the HCC related data in TCGA, ICGC, and GEO databases, and identified 106 genes which were probably specific genes related to the development from HBV to HCC. Furthermore, we built a relatively reliable predictive Risk score for HCC based on 11 genes and high Risk score was an unfavorable prognostic factor for HCC.

In HCC, HBV infection and high virus load has been widely considered the risk factors [28]. Whereas, the potential influence of HBV infection on the progression or prognosis of HCC largely remains unclear. Therefore, firstly, we have identified the possible HBV-positive HCC related specific genes. Basing on the data in GEO database, we have identified 614 DEGs and 680 DEGs in normal liver samples vs. HBV-positive liver samples and HBV-positive adjacent samples vs. HBV-positive HCC samples, respectively. Among that, 106 overlapped DEGs were probably HBV-positive HCC related specific genes, which were significantly enriched in 213 GO terms and 8 KEGG pathways. Some of these KEGG pathways have been reported in HCC previously. For example, cell cycle pathway, it has been suggested that HBV might deregulate cell cycle control to form a cellular environment conducive to infection, thereby inducing the malignant transformation of infected hepatocytes [29]. Moreover, HCC cell growth might be inhibited by inducing cell cycle arrest and apoptosis [30]. Moreover, several well known tumor related pathways were also observed, such as TNF signaling pathway, p53 signaling pathway, IL-17 signaling pathway, and so on. Among them, some genes/ lncRNAs/ proteins were evidenced to involve in the regulation of proliferation or metastasis of HCC via TNF signaling pathway [31,32,33]. A recent report has demonstrated the potential important role of p53 signaling pathway in the development of HBV-related HCC [34], which was also support our notion indirectly. On the other hand, based on these 106 DEGs, all HCC samples in TCGA could be divided in 4 clusters with different prognosis, and most HBV-positive HCC patients (74.6%) had worse prognosis. Our findings implied the importance of these 106 HBV-positive HCC related specific genes.

Subsequently, univariate Cox and LASSO Cox regression analysis were conducted on these 106 genes and the HCC data in TCGA, then 11 optimal genes were selected to build a Risk score, including HMMR, MCM6, TPX2, KIF20A, CCL20, RGS2, NUSAP1, FABP5, FZD6, PBK, and STK39. High risk HCC patients were evidenced to have worse OS in both training set and validation set. Moreover, Risk score was an independent prognostic factor for HCC. We also found some clues of the optimal genes to indirectly support our prognostic model. HMMR was found to be dysregulated in HBV related HCC [35], besides HMMR was also identified as a candidate gene involving the mechanisms behind HCC in China [36]. Whereas, the prognostic value or exact role HMMR has been seldom studied in HBV related HCC, which still needs to be clarified then. MCM6 has been suggested as a potential prognostic biomarker for HCC [37]. Moreover, MCM6 was also found to play a vital role in the progression of HCC in Chinese Zhuang population [38]. These studies were both in line with our data. TPX2 has been associated with the carcinogenesis and proliferation of HBV-related HCC [39, 40], while more details are not clear. KIF20A was reported to be related to the OS of HCC patients [41], besides, a prognostic marker based on 12 genes included KIF20A showed good predictive effect [42], both of which supported our results. High expression of CCL20 has been documented to be correlated with the poor prognosis of HCC patients [43]. Moreover, NUSAP1 [44], FABP5 [45], PBK [46], and STK39 [47] have been indicated to associate with the progression, metastasis, invasion, or prognosis of HCC directly or indirectly, which provided more evidence of our Risk score. Despite few studies of RGS2 and FZD6 were found in HCC, which deserved more exploration in the future. All above data evidenced that our Risk score was a relatively reliable prognostic predictive tool for HCC. Additionally, our Nomogram based on Risk score and stage had a good performance, which might make our Risk score more convincing.


In conclusion, via our joint analyses preformed on the HCC related data downloaded from three public databases, we have firstly revealed a prognostic signature based on HBV related specific genes in HCC. The Risk score constructed basing on 11 genes has a good prognostic predictive performance, and high Risk score is a poor prognostic indicator.

Availability of data and materials

The datasets generated and analysed during the current study are available in The Cancer Genome Atlas (TCGA,, International Cancer Genome Consortium (ICGC,, and Gene Expression Omnibus (GEO,, accession number: GSE83148 and GSE121248).



Hepatocellular carcinoma


Hepatitis B virus


Hepatitis C virus


Mutation annotation format


The cancer genome Atlas


International cancer genome consortium


Gene expression omnibus


Differentially expressed genes


Gene ontology


Kyoto encyclopedia of genes and genomes


Overall survival


  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and Mortality Worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49.

    Article  Google Scholar 

  2. Huang J, Lok V, Ngai CH, Chu C, Patel HK, Thoguluva Chandraseka V, et al. Disease burden, risk factors, and recent trends of liver cancer: a global country-level analysis. Liver Cancer. 2021;10(4):330–45.

    Article  CAS  Google Scholar 

  3. Fujiwara N, Friedman SL, Goossens N, Hoshida Y. Risk factors and prevention of hepatocellular carcinoma in the era of precision medicine. J Hepatol. 2018;68(3):526–49.

    Article  Google Scholar 

  4. Makarova-Rusher OV, Altekruse SF, McNeel TS, Ulahannan S, Duffy AG, Graubard BI, et al. Population attributable fractions of risk factors for hepatocellular carcinoma in the United States. Cancer. 2016;122(11):1757–65.

    Article  Google Scholar 

  5. Zhang BH, Yang BH, Tang ZY. Randomized controlled trial of screening for hepatocellular carcinoma. J Cancer Res Clin Oncol. 2004;130(7):417–22.

    Article  Google Scholar 

  6. Huang J, Yan L, Cheng Z, Wu H, Du L, Wang J, et al. A randomized trial comparing radiofrequency ablation and surgical resection for HCC conforming to the Milan criteria. Ann Surg. 2010;252(6):903–12.

    Article  Google Scholar 

  7. Hirao A, Sato Y, Tanaka H, Nishida K, Tomonari T, Hirata M, et al. MiR-125b-5p is involved in Sorafenib Resistance through Ataxin-1-Mediated epithelial-mesenchymal transition in Hepatocellular Carcinoma. Cancers (Basel). 2021;13:19.

    Article  Google Scholar 

  8. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008;359(4):378–90.

    Article  CAS  Google Scholar 

  9. Chen ZH, Zhang XP, Lu YG, Li LQ, Chen MS, Wen TF, et al. Actual long-term survival in HCC patients with portal vein tumor thrombus after liver resection: a nationwide study. Hepatol Int. 2020;14(5):754–64.

    Article  Google Scholar 

  10. Chidambaranathan-Reghupaty S, Fisher PB, Sarkar D. Hepatocellular carcinoma (HCC): epidemiology, etiology and molecular classification. Adv Cancer Res. 2021;149:1–61.

    Article  Google Scholar 

  11. Zhang B, Tang B, Gao J, Li J, Kong L, Qin L. A hypoxia-related signature for clinically predicting diagnosis, prognosis and immune microenvironment of hepatocellular carcinoma patients. J Transl Med. 2020;18(1):342.

    Article  CAS  Google Scholar 

  12. Hu B, Yang XB, Sang XT. Development and verification of the hypoxia-related and immune-associated prognosis signature for hepatocellular carcinoma. J Hepatocell Carcinoma. 2020;7:315–30.

    Article  CAS  Google Scholar 

  13. Villanueva A. Hepatocellular Carcinoma. N Engl J Med. 2019;380(15):1450–62.

    Article  CAS  Google Scholar 

  14. Liu J, Liang W, Jing W, Liu M. Countdown to 2030: eliminating hepatitis B disease, China. Bull World Health Organ. 2019;97(3):230–8.

    Article  Google Scholar 

  15. Maucort-Boulch D, de Martel C, Franceschi S, Plummer M. Fraction and incidence of liver cancer attributable to hepatitis B and C viruses worldwide. Int J Cancer. 2018;142(12):2471–7.

    Article  CAS  Google Scholar 

  16. Zhao X, Sun L, Mu T, Yi J, Ma C, Xie H, et al. An HBV-encoded miRNA activates innate immunity to restrict HBV replication. J Mol Cell Biol. 2020;12(4):263–76.

    Article  CAS  Google Scholar 

  17. El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. 2012;142(6):1264–73 e1261.

    Article  Google Scholar 

  18. Jia L, Gao Y, He Y, Hooper JD, Yang P. HBV induced hepatocellular carcinoma and related potential immunotherapy. Pharmacol Res. 2020;159:104992.

    Article  CAS  Google Scholar 

  19. Rehermann B, Thimme R. Insights from antiviral therapy into immune responses to hepatitis B and C virus infection. Gastroenterology. 2019;156(2):369–83.

    Article  CAS  Google Scholar 

  20. Gao Q, Zhu H, Dong L, Shi W, Chen R, Song Z, et al. Integrated proteogenomic characterization of HBV-related hepatocellular carcinoma. Cell. 2019;179(2):561-77 e522.

    Article  CAS  Google Scholar 

  21. Yan Y, Lu Y, Mao K, Zhang M, Liu H, Zhou Q, et al. Identification and validation of a prognostic four-genes signature for hepatocellular carcinoma: integrated ceRNA network analysis. Hepatol Int. 2019;13(5):618–30.

    Article  Google Scholar 

  22. Fang Q, Chen H. The significance of m6A RNA methylation regulators in predicting the prognosis and clinical course of HBV-related hepatocellular carcinoma. Mol Med. 2020;26(1):60.

    Article  Google Scholar 

  23. Friedman J, Hastie T, Tibshirani R. Regularization paths for generalized linear models via coordinate descent. J Stat Softw. 2010;33(1):1–22.

    Article  Google Scholar 

  24. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47.

    Article  Google Scholar 

  25. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16(5):284–7.

    Article  CAS  Google Scholar 

  26. Newman AM, Liu CL, Green MR, Gentles AJ, Feng W, Xu Y, et al. Robust enumeration of cell subsets from tissue expression profiles. Nat Methods. 2015;12(5):453–7.

    Article  CAS  Google Scholar 

  27. Liu W, Zhou JG, Sun Y, Zhang L, Xing BC. Hepatic resection improved the long-term survival of patients with BCLC stage B hepatocellular carcinoma in Asia: a systematic review and meta-analysis. J Gastrointest Surg. 2015;19(7):1271–80.

    Article  Google Scholar 

  28. Ma S, Qin K, Ouyang H, Zhu H, Lei P, Shen G. HBV infection exacerbates PTEN defects in hepatocellular carcinoma through upregulation of miR-181a/382/362/19a. Am J Transl Res. 2020;12(7):3780–91.

    CAS  Google Scholar 

  29. Xia Y, Cheng X, Li Y, Valdez K, Chen W, Liang TJ. Hepatitis B virus deregulates the cell cycle to promote viral replication and a premalignant phenotype. J Virol. 2018;92(19):e00722-18.

    Article  Google Scholar 

  30. Chen SY, Chao CN, Huang HY, Fang CY. Flavopereirine inhibits hepatocellular carcinoma cell growth by inducing cell-cycle arrest, apoptosis, and autophagy-related protein expression. Anticancer Res. 2020;40(12):6907–14.

    Article  CAS  Google Scholar 

  31. Zhang GP, Yue X, Li SQ, Cathepsin C. Interacts with TNF-alpha/p38 MAPK signaling pathway to promote proliferation and metastasis in Hepatocellular Carcinoma. Cancer Res Treat. 2020;52(1):10–23.

    Article  CAS  Google Scholar 

  32. Ma W, Chen X, Wu X, Li J, Mei C, Jing W, et al. Long noncoding RNA SPRY4-IT1 promotes proliferation and metastasis of hepatocellular carcinoma via mediating TNF signaling pathway. J Cell Physiol. 2020;235(11):7849–62.

    Article  CAS  Google Scholar 

  33. Nagasawa T, Matsushima-Nishiwaki R, Yasuda E, Matsuura J, Toyoda H, Kaneoka Y, et al. Heat shock protein 20 (HSPB6) regulates TNF-alpha-induced intracellular signaling pathway in human hepatocellular carcinoma cells. Arch Biochem Biophys. 2015;565:1–8.

    Article  CAS  Google Scholar 

  34. Yu M, Xu W, Jie Y, Pang J, Huang S, Cao J, et al. Identification and validation of three core genes in p53 signaling pathway in hepatitis B virus-related hepatocellular carcinoma. World J Surg Oncol. 2021;19(1):66.

    Article  Google Scholar 

  35. Sha M, Cao J, Zong ZP, Xu N, Zhang JJ, Tong Y, et al. Identification of genes predicting unfavorable prognosis in hepatitis B virus-associated hepatocellular carcinoma. Ann Transl Med. 2021;9(12):975.

    Article  CAS  Google Scholar 

  36. Zhang P, Feng J, Wu X, Chu W, Zhang Y, Li P. Bioinformatics Analysis of candidate genes and pathways related to hepatocellular carcinoma in China: a study based on public databases. Pathol Oncol Res. 2021;27:588532.

    Article  Google Scholar 

  37. Liao X, Liu X, Yang C, Wang X, Yu T, Han C, et al. Distinct diagnostic and prognostic values of minichromosome maintenance gene expression in patients with hepatocellular carcinoma. J Cancer. 2018;9(13):2357–73.

    Article  Google Scholar 

  38. Jia W, Xie L, Wang X, Zhang Q, Wei B, Li H, et al. The impact of MCM6 on hepatocellular carcinoma in a Southern Chinese Zhuang population. Biomed Pharmacother. 2020;127:110171.

    Article  CAS  Google Scholar 

  39. Zeng XC, Zhang L, Liao WJ, Ao L, Lin ZM, Kang W, et al. Screening and identification of potential biomarkers in hepatitis B virus-related hepatocellular carcinoma by bioinformatics analysis. Front Genet. 2020;11:555537.

    Article  CAS  Google Scholar 

  40. Wang Y, Wang H, Yan Z, Li G, Hu G, Zhang H, et al. The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation. Cell Commun Signal. 2020;18(1):116.

    Article  Google Scholar 

  41. Chen X, Liao L, Li Y, Huang H, Huang Q, Deng S. Screening and functional prediction of key candidate genes in hepatitis B virus-associated hepatocellular carcinoma. Biomed Res Int. 2020;2020:7653506.

    Google Scholar 

  42. Ouyang G, Yi B, Pan G, Chen X. A robust twelve-gene signature for prognosis prediction of hepatocellular carcinoma. Cancer Cell Int. 2020;20:207.

    Article  CAS  Google Scholar 

  43. Ding X, Wang K, Wang H, Zhang G, Liu Y, Yang Q, et al. High expression of CCL20 is associated with poor prognosis in patients with hepatocellular carcinoma after curative resection. J Gastrointest Surg. 2012;16(4):828–36.

    Article  Google Scholar 

  44. Yang Z, Li J, Feng G, Wang Y, Yang G, Liu Y, et al. Hepatitis B virus X protein enhances hepatocarcinogenesis by depressing the targeting of NUSAP1 mRNA by miR-18b. Cancer Biol Med. 2019;16(2):276–87.

    Article  Google Scholar 

  45. Liu F, Liu W, Zhou S, Yang C, Tian M, Jia G, et al. Identification of FABP5 as an immunometabolic marker in human hepatocellular carcinoma. J Immunother Cancer. 2020.

    Article  Google Scholar 

  46. Yang QX, Zhong S, He L, Jia XJ, Tang H, Cheng ST, et al. PBK overexpression promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR signaling pathway. Cancer Lett. 2019;452:90–102.

    Article  CAS  Google Scholar 

  47. Chen J, Zhou L, Yang J, Xie H, Liu L, Li Y. Knockdown of STK39 suppressed cell proliferation, migration, and invasion in hepatocellular carcinoma by repressing the phosphorylation of mitogen-activated protein kinase p38. Bioengineered. 2021;12(1):6529–37.

    Article  CAS  Google Scholar 

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This work was supported by the Tianjin Natural Science Foundation (Grant No.19JCQNJC08700); the Educational Committee Foundation of Tianjin City (Grant No.2020KJ196) and the Foundation of Logistics University of People’s Armed Police Force (Grant No.WHJ202111).

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Conceptualization and Visualization: LW and MMQ. Administrative support and Writing—review & editing: BY. Data curation and Formal analysis: LLW, ZXL, XYM and LH. Writing—original draft: All authors. All authors read and approved the final manuscript to be published.

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Correspondence to Bing Yang.

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Additional file 1

. Table S1 The 106 overlapped DEGs.

Additional file 2

. Table S2 The detailed functional enrichment results.

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Wang, L., Qiu, M., Wu, L. et al. Construction and validation of prognostic signature for hepatocellular carcinoma basing on hepatitis B virus related specific genes. Infect Agents Cancer 17, 60 (2022).

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