Identification and validation of an H2AZ1-based index model: a novel prognostic tool for hepatocellular carcinoma.

The H2A.Z variant histone 1 (H2AZ1) is aberrantly expressed in various tumors, correlating with an unfavorable prognosis. However, its role in hepatocellular carcinoma (HCC) remains unclear. We aimed to elucidate the pathways affected by H2AZ1 and identify promising therapeutic targets for HCC. Following bioinformatic analysis of gene expression and clinical data from The Cancer Genome Atlas and Gene Expression Omnibus database, we found 6,344 dysregulated genes related to H2AZ1 overexpression in HCC tissues (P < 0.05). We performed weighted gene co-expression network analysis to identify the gene module most related to H2AZ1. The H2AZ1-based index was further developed using Cox regression analysis, which revealed that the poor prognosis in the high H2AZ1-based index group could be attributed to elevated tumor stemness (P < 0.05). Moreover, the clinical model showed good prognostic potential (AUC > 0.7). We found that H2AZ1 knockdown led to reduced superoxide dismutase (SOD) activity, elevated malondialdehyde (MDA) levels, and increased apoptosis rate in tumor cells (P < 0.001). Thus, we developed an H2AZ1-based index model with the potential to predict the prognosis of patients with HCC. Our findings provide initial evidence that H2AZ1 overexpression plays a pivotal role in HCC initiation and progression.

Genetic association of cardiovascular disease related biomarkers with the overall survival of hepatocellular carcinoma: a Mendelian randomization analysis.

Observational studies have reported associations between circulating biomarkers related to cardiovascular disease and the survival of patients with hepatocellular carcinoma. However, the relationship between these biomarkers and survival remains controversial. We conducted a two-sample Mendelian randomization analysis to investigate possible causal associations between cardiovascular disease biomarkers and hepatocellular carcinoma survival. Genetic risk scores, calculated using individual-level data from 866 cases of hepatitis B virus-related hepatocellular carcinoma in Guangxi, were utilized as proxies for four cardiovascular disease biomarkers: C-reactive protein, Apolipoprotein A-1, Cystatin C, and Lipoprotein(a). Associations between the genetic scores and survival were analyzed using Cox regression. The inverse-variance weighted method was used to estimate the summary statistics for the biomarkers and survival. Considering the multiple comparisons, the statistical significance was set at P < 0.0125. We observed a significant risk signal between genetically increased Cystatin C levels and poorer survival in hepatocellular carcinoma (HR for genetic scores = 1.29, 95% CI = 1.02-1.64; HR for inverse-variance weighted = 2.60, 95% CI = 1.45-4.65). Furthermore, we found a causal relationship of genetically determined Cystatin C and Lipoprotein(a) level with the survival of hepatocellular carcinoma patients with embolus. Our findings indicated the causal effects of increased levels of Cystatin C and Lipoprotein(a) on poorer survival in hepatocellular carcinoma.

Potentially functional genetic variants in RPS6KA4 and MAP2K5 in the MAPK signaling pathway predict HBV-related hepatocellular carcinoma survival.

Hepatocellular carcinoma (HCC) ranks the third leading cause of cancer deaths with a dismal 5-year survival rate. The mitogen-activated protein kinase (MAPK) signaling pathway is abnormally activated in HCC to promote growth and aggressive metastatic potential of cancer cells. Therefore, genetic variants in the MAPK signaling pathway may serve as potential predictors of Hepatitis B virus (HBV)-related HCC survival. In the present study, we performed a two-stage survival analysis to evaluate the associations between 10,912 single nucleotide polymorphisms (SNPs) in 79 MAPK signaling pathway genes and the overall survival (OS) of 866 HBV-related HCC patients, followed by functional annotation. In combined datasets, we identified two novel and potential functional SNPs (RPS6KA4 rs600377 T>G and MAP2K5 rs17300363 A>C) as prognostic factors for HBV-related HCC, with adjusted allelic hazards ratios of 1.24 (95% confidence interval [CI] = 1.05-1.46, p = 0.010) and 1.48 (1.15-1.91, p = 0.001), respectively. Furthermore, their combined risk genotypes also predicted a poor survival in a dose-response manner in the combined data set (Ptrend < 0.001). Additional functional analysis showed that RPS6KA4 rs600377 G and MAP2K5 rs17300363 C alleles were associated with elevated mRNA expression levels of the corresponding genes in normal tissues. These results provide new insights into the role of genetic variants in the MAPK signaling pathway genes in HBV-related HCC survival.

Genetic variants of SOS2, MAP2K1 and RASGRF2 in the RAS pathway genes predict survival of HBV-related hepatocellular carcinoma patients.

The RAS pathway participates in the cascade of proliferation and cell division process, and the activated RAS pathway can lead to tumorigenesis including hepatocellular carcinoma (HCC). However, few studies have explored the effects of genetic variants in the RAS pathway-related genes on the survival of patients with HBV-related HCC. In the present study, we assessed the associations between 11,658 single-nucleotide polymorphisms (SNPs) in 62 RAS pathway genes and the overall survival (OS) of 866 HBV-related HCC individuals, which were randomly split (1:1) into discovery and validation datasets. As a result, three potentially functional SNPs were identified, based on multivariable cox proportional hazards regression analyses, in SOS Ras/Rho guanine nucleotide exchange factor 2 (SOS2, rs4632055 A > G), Ras protein-specific guanine nucleotide releasing factor 2 (RASGRF2, rs26418A > G) and mitogen-activated protein kinase 1 (MAP2K1,rs57120695 C > T), which were significantly and independently associated with OS of HBV-related HCC patients [adjusted hazards ratios (HRs) of 1.42, 1.32 and 1.50, respectively; 95% confidence intervals (CI), 1.14 to 1.76, 1.15 to 1.53 and 1.15 to 1.97, respectively; P = 0.001, < 0.001 and 0.003, respectively]. Additionally, the joint effects as the unfavorable genotypes of these three SNPs showed a significant association with the poor survival of HCC (trend test P < 0.001). The expression quantitative trait loci (eQTL) analysis further revealed that the rs4632055 G allele and the rs26418 A allele were associated with lower mRNA expression levels of SOS2 and RASGRF2, respectively. Collectively, these potentially functional SNPs of RASGRF2, SOS2 and M2PAK1 may become potential prognostic biomarkers for HBV-related HCC after hepatectomy.

A novel signature constructed by super-enhancer-related genes for the prediction of prognosis in hepatocellular carcinoma and associated with immune infiltration.

Background: Super-enhancer (SE) refers to a regulatory element with super transcriptional activity, which can enrich transcription factors and drive gene expression. SE-related genes play an important role in the pathogenesis of malignant tumors, including hepatocellular carcinoma (HCC). Methods: The SE-related genes were obtained from the human super-enhancer database (SEdb). Data from the transcriptome analysis and related clinical information with HCC were obtained from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) database. The upregulated SE-related genes from TCGA-LIHC were identified by the DESeq2R package. Multivariate Cox regression analysis was used to construct a four-gene prognostic signature. According to the median risk score, HCC patients were divided into high-risk and low-risk group patients. Results: The Kaplan-Meier (KM) curve showed that a significantly worse prognosis was found for the high-risk group (P<0.001). In the TCGA-LIHC dataset, the area under the curve (AUC) values were 0.737, 0.662, and 0.667 for the model predicting overall survival (OS) over 1-, 3-, and 5- years, respectively, indicating the good prediction ability of our prediction model. This model's prognostic value was further validated in the LIRI-JP dataset and HCC samples (n=65). Furthermore, we found that higher infiltration level of M0 macrophages and upregulated of CTLA4 and PD1 in the high-risk group, implying that immunotherapy could be effective for those patients. Conclusion: These results provide further evidence that the unique SE-related gene model could accurately predict the prognosis of HCC.

Clinical Implication of E2F Transcription Factor 1 in Hepatocellular Carcinoma Tissues.

Background: To date, the clinical management of advanced hepatocellular carcinoma (HCC) patients remains challenging and the mechanisms of E2F transcription factor 1 (E2F1) underlying HCC are obscure. Materials and Methods: Our study integrated datasets mined from several public databases to comprehensively understand the deregulated expression status of E2F1. Tissue microarrays and immunohistochemistry staining was used to validate E2F1 expression level. The prognostic value of E2F1 was assessed. In-depth subgroup analyses were implemented to compare the differentially expressed levels of E2F1 in HCC patients with various tumor stages. Functional enrichments were used to address the predominant targets of E2F1 and shedding light on their potential roles in HCC. Results: We confirmed the elevated expression of E2F1 in HCC. Subgroup analyses indicated that elevated E2F1 level was independent of various stages in HCC. E2F1 possessed moderate discriminatory capability in differentiating HCC patients from non-HCC controls. Elevated E2F1 correlated with Asian race, tumor classification, neoplasm histologic grade, eastern cancer oncology group, and plasma AFP levels. Furthermore, high E2F1 correlated with poor survival condition and pooled HR signified E2F1 as a risk factor for HCC. Enrichment analysis of differentially expressed genes, coexpressed genes, and putative targets of E2F1 emphasized the importance of cell cycle pathway, where CCNE1 and CCNA2 served as hub genes. Conclusions: We confirmed the upregulation of E2F1 and explored the prognostic value of E2F1 in HCC patients. Two putative targeted genes (CCNE1 and CCNA2) of E2F1 were identified for their potential roles in regulating cell cycle and promote antiapoptotic activity in HCC patients.

A functional variant in the RAD51 3' UTR is associated with survival of hepatocellular carcinoma patients.

The RAD51 gene plays an important role in DNA repair by homologous recombination, and is involved in the development and progression of multiple cancers. Single nucleotide polymorphisms in RAD51 have been previously described to impact the prognosis of patients with cancers, however, it is still unclear whether this is also true for hepatocellular carcinoma (HCC). This study therefore aimed to identify genetic variants in RAD51 and determine the effect on the survival of patients with HCC. In this study, we performed genotyping assays for RAD51 polymorphisms in a cohort of 368 patients with HCC who had underwent hepatectomy. Using multivariate cox proportional hazards model and Kaplan-Meier analyses with log-rank tests, we compared the survival of patients with HCC according to RAD51 SNP genotypes. We identified one potential functional variant, rs12593359, located in a microRNA (miRNA) binding site in the RAD51 3' untranslated region, to be an independent predictor of overall survival of patients with HCC in the dominant model. Patients carrying GT/TT genotypes had a significantly increased risk of death when compared with those carrying GG genotype (adjusted hazard ratio = 1.34, 95 % confidence interval = 1.02-1.76, P = 0.035). Kaplan-Meier curve analysis showed a markedly shorter survival time for patients with HCC carrying GT/TT genotypes of SNP rs12593359 (19.0 months vs 36.0 months; Plog-rank = 0.012). Notably, this effect was particularly pronounced in several subgroups of patients (e.g., males, Hepatitis B virus-positive patients, patients with a single tumor nodule, patients with alpha-fetoprotein (AFP) < 400 ng/ml, or patients who were cancer embolus-free). Additional expression analysis of quantitative trait loci showed that SNP rs12593359 was significantly associated with RAD51 mRNA expression levels in 483 cell-cultured fibroblasts (P = 1.1 × 10-4). These findings provide evidence that RAD51 rs12593359 is associated with HCC survival and may serve as a promising predictor of survival in patients with HCC.

A novel risk score based on immune-related genes for hepatocellular carcinoma as a reliable prognostic biomarker and correlated with immune infiltration.

Background: Immunological-related genes (IRGs) play a critical role in the immune microenvironment of tumors. Our study aimed to develop an IRG-based survival prediction model for hepatocellular carcinoma (HCC) patients and to investigate the impact of IRGs on the immune microenvironment. Methods: Differentially expressed IRGs were obtained from The Genomic Data Commons Data Portal (TCGA) and the immunology database and analysis portal (ImmPort). The univariate Cox regression was used to identify the IRGs linked to overall survival (OS), and a Lasso-regularized Cox proportional hazard model was constructed. The International Cancer Genome Consortium (ICGC) database was used to verify the prediction model. ESTIMATE and CIBERSORT were used to estimate immune cell infiltration in the tumor immune microenvironment (TIME). RNA sequencing was performed on HCC tissue specimens to confirm mRNA expression. Results: A total of 401 differentially expressed IRGs were identified, and 63 IRGs were found related to OS on the 237 up-regulated IRGs by univariate Cox regression analyses. Finally, five IRGs were selected by the LASSO Cox model, including SPP1, BIRC5, STC2, GLP1R, and RAET1E. This prognostic model demonstrated satisfactory predictive value in the ICGC dataset. The risk score was an independent predictive predictor for OS in HCC patients. Immune-related analysis showed that the immune infiltration level in the high-risk group was higher, suggesting that the 5-IRG signature may play an important role in mediating immune escape and immune resistance in the TIME of HCC. Finally, we confirmed the 5-IRG signature is highly expressed in 65 HCC patients with good predictive power. Conclusion: We established and verified a new prognosis model for HCC patients based on survival-related IRGs, and the signature could provide new insights into the prognosis of HCC.

Potentially functional variants of MAP3K14 in the NF-κB signaling pathway genes predict survival of HBV-related hepatocellular carcinoma patients.

Background: The NF-κB signaling pathway plays an important role in associating inflammation with tumor development and progression. However, few studies have reported that roles of genetic variants of the NF-κB signaling pathway genes in survival of patients with HBV-related hepatocellular carcinoma (HBV-HCC), especially with regards to potentially functional SNPs. Methods: We used multivariate Cox proportional hazards regression to evaluate associations between 2,060 single nucleotide polymorphisms (SNPs) in 20 NF-κB signaling pathway genes and survival of 866 HBV-HCC patients, which were randomly split (1:1) into discovery and validation datasets. Expression quantitative trait loci (eQTL) analysis was conducted to identify associations between survival-associated SNPs and mRNA expression of corresponding genes. Furthermore, online database was used to assess mRNA expression of corresponding genes and survival. Finally, receiver operating characteristic (ROC) curves were used to assess the prediction accuracy of models integrating both clinical and genetic variables on HCC survival. Results: A total of 6 SNPs in MAP3K14 remained significantly associated with OS of HBV-HCC patients (P<0.05, BFDP<0.8). Further eQTL analysis demonstrated that significant correlations between the rs2074292 (G>A) A allele was associated with higher mRNA expression levels of MAP3K14 (P=0.044) in normal liver tissue, which was associated with worse survival of HBV-HCC patients. In the additive model, after adjusting for age, sex, smoking status, drinking status, AFP level, cirrhosis, embolus and BCLC stage, the combined dataset showed that HBV-HCC patients carrying the rs2074292 AA and GA genotypes (HR=1.71, 95%CI= 1.29-2.27, P=0.000) (HR=1.40, 95%CI=1.10-1.77, P=0.005) have worse OS than GG genotype, respectively. The addition of risk genotypes to the prediction models increased the AUC significantly from 71.15% to 73.11% (P=0.012) and from 72.55% to 74.21% (P=0.010) for 1-year and 3-year OS, respectively. Conclusion: Our study indicated that MAP3K14 rs2074292 A allele may be a potential predictor of HBV-HCC survival, likely regulating MAP3K14 mRNA expression.