Clinical Prognosis Of Hepatocellular Carcinoma Research Articles

A disulfidptosis-related lncRNA signature for analyzing tumor microenvironment and clinical prognosis in hepatocellular carcinoma.

Disulfidptosis is a recently identified form of non-apoptotic programmed cell death which distinguishes itself from classical cell death pathways. However, the prognostic implications of disulfidptosis-related long non-coding RNAs (DRLs) and their underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unexplored. In this study, we leveraged RNA-sequencing data and clinical information of HCC patients from the TCGA database. Through expression correlation and prognostic correlation analyses, we identified a set of top-performing long non-coding RNAs. Subsequently, a 5-DRLs predictive signature was established by conducting a Lasso regression analysis. This signature effectively stratified patients into high- and low-risk groups, revealing notable differences in survival outcomes. Further validation through univariate and multivariate Cox regression analyses confirmed that the risk score derived from our signature independently predicted the prognosis of HCC patients. Moreover, we observed significant disparities in immune cell infiltration and tumor mutation burden (TMB) between the two risk groups, shedding light on the potential connection between immune-related mechanisms and disulfidptosis. Notably, the signature also exhibited predictive value in the context of chemotherapeutic drug sensitivity and immunotherapy efficacy for HCC patients. Finally, we performed experimental validation at both cellular and patient levels and successfully induced a disulfidptosis phenotype in HCC cells. In general, this multifaceted approach provides a comprehensive overview of DRLs profiles in HCC, culminating in the establishment of a novel risk signature that holds promise for predicting prognosis and therapy outcomes of HCC patients.

LAYN Serves as a Prognostic Biomarker and Downregulates Tumor-Infiltrating CD8+ T Cell Function in Hepatocellular Carcinoma.

Layilin (LAYN) represents a valuable prognostic biomarker across various tumor types, while also serving as an innovative indicator of dysfunctional or exhausted CD8+ T cells and exhibiting correlation with immune context. However, the immune function and prognostic significance of LAYN in hepatocellular carcinoma (HCC) remain unexplored. Therefore, our objective is to investigate the role of LAYN in CD8+ T cell exhaustion, clinical prognosis, and the tumor microenvironment within HCC. TIMER or GEPIA databases were used to analyze LAYN expression level and its correlation with immune infiltration in HCC. Bioinformatics analysis was conducted on TCGA and scRNA-seq cohorts. The evaluation of LAYN expression level in fresh specimens was performed through IF, IHC, and ELISA assays. Flow cytometry and mRNA-seq were employed to investigate co-expressed genes of LAYN, the LAYN+CD8+ T cell exhaustion signature and immune function. Cell proliferation ability and killing activity were assessed using CCK8 and CFSE/PI. The expression level of LAYN in HCC tumors was significantly higher compared to peri-tumors. Patients with high levels of LAYN exhibited poorer OS. GO or KEGG analysis confirmed that LAYN was involved in immune response and was positively associated with CD8+ T cell immune infiltration levels. Furthermore, LAYN negatively regulated the immune function of CD8+ T cells, leading to dysfunctional phenotypes characterized by elevated levels of CD39, TIM3 and reduced levels of perforin, TNF-α, Ki-67. CFSE/PI assays demonstrated that LAYN+CD8+ T cells displayed decreased cytotoxic activity. Additionally, there was a positive correlation between LAYN and CD146 levels, which are involved in adhesion and localization processes of CD8+ T cells. Interestingly, blocking LAYN partially restored the exhaustion properties of CD8+ T cells. LAYN exhibits a strong correlation with immune infiltration in the TME and represents a novel biomarker for predicting clinical prognosis in HCC. Moreover, targeting LAYN may hold promise as an effective strategy for HCC immunotherapy.

Cullin-associated and neddylation-dissociated 1 regulate reprogramming of lipid metabolism through SKP1-Cullin-1-F-boxFBXO11 -mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma.

Enhanced de novo lipogenesis is essential for hepatocellular carcinoma (HCC). Abnormally high cullin-associated and neddylation-dissociated 1 (CAND1) expression is associated with poor clinical prognosis in HCC. The SKP1-Cullin-1-F-box (SCF) complex consists of the SKP1, Cullin-1 and F-box proteins (FBPs) and performs multiple functions including adipogenesis. SCF complex was modulated by CAND1, but Whether and how the CAND1 promotes HCC by regulating SCF complex and lipogenesis are unknown. HCC samples were used to analyze the correlations between CAND1 expression and clinicopathological characteristics such as survival and prognosis. The in vitro functions of CAND1, FBXO11 and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) were measured by cell proliferation, colony formation and migration assays. The in vivo functions were tested in multiple mouse liver cancer models including patient-derived xenograft (PDX), cell line-derived xenograft and AKT/NRASV12-induced primary liver cancer models. Injections of adeno-associated virus targeting CAND1 (AAV-shCAND1) were performed to evaluate the therapeutic efficacy of targeting CAND1. RNA-Seq and lipidomic assays followed by serial biochemical experiments including mass spectrometry, immunoprecipitation and GST pull-down were performed to dissect the underlying mechanisms. CAND1 promoted the expression of lipid synthesis genes by disrupting SCF complex assembly and lipid accumulation. Furthermore, we identified hnRNPA2B1 as a novel F-box protein 11 (FBXO11)-binding partner. FBXO11 directly bound to hnRNPA2B1 and promoted hnRNPA2B1 ubiquitination and subsequent degradation. Our evaluations of the therapeutic efficacy of AAV-shCAND1 injections confirmed that targeting the CAND1-SCFFBXO11 -hnRNPA2B1A signalling axis was therapeutically effective. CAND1 downregulation significantly reduced the tumour burden in a primary mouse liver cancer model and a PDX model. Our results highlight that CAND1 is associated with poor prognosis in HCC and regulates lipid metabolic reprogramming by dissociating the SCF complex. Targeting the CAND1-SCFFBXO11 -hnRNPA2B1 axis may be a novel strategy for HCC treatment.

Six Cell Cycle-related Genes Serve as Potential Prognostic Biomarkers and Correlated with Immune Infiltrates in Hepatocellular Carcinoma.

Background: Cell cycle-related genes (CDK1, CDK5, CDC20, CCNA2, CCNB1, and CCNB2) play important roles in the regulation of mitotic cell cycle in eukaryotes. However, the correlation between cell cycle-related genes and tumor-infiltrating and prognosis of hepatocellular carcinoma (HCC) needs further investigation. Methods: Two public websites, Tumor Immune Estimate Resource (TIMER) and Oncomine, were used to assess the expression levels of cycle-related genes. We also analyzed the protein expression levels of six cell cycle-related genes using the HPA database. In addition, Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA) database were used to investigate the impact of cell cycle-related gene expression levels on the clinical prognosis of HCC. The correlation between cell cycle-related genes and cancer immune infiltrates was analyzed through TIMER site. Subsequently, GEPIA and TIMER database were used to assess the correlation between the expression of six cell cycle-related genes and polygenic markers in monocytes and macrophages, respectively. The cell cycle-related genes were also analyzed to find the associated genes with the highest alteration frequency, by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) approaches of Metascape and String database, respectively. Results: The expression levels of cell cycle-related genes were up-regulated in tumor tissues compared with normal tissues. Subsequently, the expression of high cell cycle-related genes was positively correlated with poor overall survival (OS) and progression-free survival (PFS) in HCC, for CDK1 (OS: HR = 2.15, P = 1.1E-05 PFS: HR = 2.03, P = 2.3E-06), CDK5 (OS: HR = 1.85, P = 0.0035 PFS: HR = 1.26, P = 0.17), CDC20 (OS: HR = 2.49, P = 5.1E-07 PFS: HR = 1.77, P = 0.00012), CCNA2 (OS: HR = 1.92, P = 0.00018 PFS: HR = 1.96, P = 5.2E-06), CCNB1 (OS: HR = 2.34, P = 3.4E-05 PFS: HR = 1.97, P = 5.3E-06), and CCNB2 (OS: HR = 1.91, P = 0.0013 PFS: HR = 1.63, P = 0.0011), respectively. Furthermore, the transcription level of cell cycle-related genes was significantly correlated with immune infiltrating levels of CD4+ T and CD8+ T cells, neutrophils, macrophages, and dendritic cells (DCs) in HCC, respectively. Amongst them, the expression levels of CDK1, CDC20, CCNA2, CCNB1 and CCNB2 manifest strongly correlated with diverse immune marker sets in HCC. Conclusions: Our results demonstrated that cell cycle-related genes played key roles in the prognosis of HCC. Meanwhile, they were significantly correlated with immune infiltrating levels of CD4+ T cells, CD8+ T cells, neutrophils, macrophages and DCs in HCC, respectively. In addition, CDK1, CDC20, CCNA2, CCNB1 and CCNB2 expressions may be involved in the regulation of monocytes and tumor-associated macrophages (TAMs) in HCC, respectively. These findings strongly suggested that cell cycle-related genes could be used as novel biomarkers for exploring the prognosis and immune cells infiltration of HCC.

Inhibition of RNaseH2A Induces Fas-Regulated Programmed Cell Death in Hepatoma Cells

Objective: To screen clinically significant potential drug targets in liver cancer and to study the function and potential molecular mechanisms of this target protein in the development of liver cancer. Methods: By using the clinical database GEPIA to find genes that are differentially expressed in liver cancer compared to normal tissues, we further screened the genes that are highly expressed in hepatocellular carcinoma and have clinical prognostic relevance. Heat maps were used to sort these genes according to their clinical prognostic relevance, so as to screen for the target gene of interest. The characteristics of target gene expression and clinical prognosis in hepatocellular carcinoma were studied. The target gene was knocked down through siRNA, and cell proliferation experiments and apoptosis experiments were used to verify the importance of the target gene in the occurrence and development of liver cancer. Finally, we elucidated the potential molecular mechanism of the target gene’s function in liver cancer based on the mutual regulatory relationship between the target gene and key apoptosis genes. Results: 1482 genes were significantly underexpressed in liver cancer, and 725 genes were significantly overexpressed in liver cancer, of which RNaseH2A was significantly overexpressed in liver cancer and had a significant clinical prognosis. Knockdown of RNaseH2A inhibited the proliferation of hepatocellular carcinoma cells and induced apoptosis. Knockdown of RNaseH2A induced the high expression of Fas, a key gene for apoptosis, and liver cancer usually features low expression of Fas. After hepatocellular carcinoma cells that were knocked down of RNaseH2A continued were subject to Fas knockdown, hepatocellular carcinoma cell proliferation and apoptosis returned to normal levels. Conclusion: The high expression of RNaseH2A regulates the low expression of Fas, a key gene for apoptosis, thereby inhibiting apoptosis, promoting cell proliferation, and participating in the development of liver cancer.

B4GALT5 high expression associated with poor prognosis of hepatocellular carcinoma

BackgroundB4GALT5 is postulated to be an important protein in sugar metabolism that catalyzes the synthesis of lactosylceramide (LacCer). However, its role in hepatocellular carcinoma (HCC) remains unknown.MethodWe characterized the expression of B4GALT5 in HCC tissue compared to normal tissue, and explored its function of B4GALT5 in HCC by enrichment analysis based on its co-expressed gene set. Next, we checked whether B4GALT5 expression is correlated to immune infiltration level and clinical prognosis in hepatocellular carcinoma. Finally, we verified the expression of B4GALT5 using clinical samples evaluated by RT-PCR, and conducted in vitro experiments with B4GALT5-knockdown HCC cells to investigate the function of B4GALT5 in the HCC cell proliferation, migration and invasion.ResultsWe found B4GALT5 mRNA and protein expression levels were significantly high in HCC tissue compared to normal tissue. The enrichment analysis of the gene sets that co-expressed with B4GALT5 showed specificity in HCC-related pathways and functions. Also, the expression pattern of B4GALT5 was significantly related to the immune infiltration level, especially CD4+ T cell and macrophage cells. B4GALT5 higher mRNA expression was associated with poor overall survival (OS) in HCC patients. Furthermore, In vitro experiments showed that depletion of B4GALT5 significantly inhibited HCC cell proliferation, migration and invasion. This study revealed the function and its mediated pathways of B4GALT5 in HCC, indicating that B4GALT5 may serve as a prognostic biomarker of HCC.

Overexpression of CISD1 Predicts Worse Survival in Hepatocarcinoma Patients.

Background Ferroptosis plays a vital role in hepatocellular carcinoma (HCC). CISD1 is known to regulate ferroptosis negatively. However, the correlations of CISD1 to prognosis in HCC and its potential mechanism remain unclear. Aim To investigate the expression level and prognostic value of CISD1 in HCC. Methods Gene expression and clinical data for 33 cancer types in TCGA were downloaded from the UCSC Xena platform. Pan-cancer analysis was performed to determine the expression profile and prognostic value of CISD1 in human cancers. GEO datasets and Human Protein Atlas (HPA) were used to verify the mRNA and protein expression levels. The influence of CISD1 on clinical prognosis in HCC was evaluated using a Kaplan-Meier plotter. The PPI network was constructed using the STRING database and Cytoscape. GO and KEGG pathways were constructed using the “clusterProfiler” R package with the FDR cutoff of 0.05. The methylation at the CISD1 promoter was detected using UALCAN and GEO datasets. The correlations between CISD1 and HCC immune infiltrates were investigated via TIMER. Results Pan-cancer analysis of TCGA data showed that CISD1 is differentially expressed in multiple tumors. Data of gene expression microarrays reveal that the mRNA expression of CISD1 is higher in HCC than that in normal tissue. The protein level of CISD1, validated by the Human Protein Atlas (HPA) database, was upregulated consistently with mRNA levels in HCC samples. High CISD1 expression was associated with better overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), and progression-free survival (PFS) in LGG, but with poorer OS, DFS, DSS, and PFS in LIHC. Protein-protein interaction (PPI) analysis and GO/KEGG analysis showed that the PPI network and GO term of CISD1 were mainly associated with energy and iron metabolism. Promoter hypomethylation correlated with overexpression of CISD1. CISD1 expression was positively correlated with infiltrating levels of CD8+ T cells, macrophages, neutrophils, and dendritic cells (DCs) in HCC. Conclusions These findings suggest that hypomethylation of the CISD1 promoter increases its expression in HCC. CISD1 is associated with prognosis and immune infiltrating levels of CD8+ T cells, macrophages, neutrophils, and DCs in HCC patients. These findings suggest that CISD1 can be used as a prognostic biomarker for determining prognosis in HCC.

Correlation analysis of RDM1 gene with immune infiltration and clinical prognosis of hepatocellular carcinoma.

Purpose: Liver hepatocellular carcinoma (LIHC) is one of the most common primary malignant liver tumors worldwide. The RAD52 motif-containing protein 1 (RDM1) has been shown to play a role in mediating DNA damage repair and homologous recombination. The present study was designed to determine the expression of RDM1 and its prognostic value as well as its relationship with immune infiltration in LIHC patients.Methods: Oncomine and Tumor Immunoassay Resource were used to assess the expression of RDM1. PrognoScan and Kaplan–Meier bioinformatics database were used to analyze the impact of clinical influencing factors on prognosis. Finally, the Tumor Immune Assessment Resource (TIMER) and Gene Expression Analysis Interactive Analysis (GEPIA) databases were used to detect the correlation between the expression of RDM1 and expression of marker genes related to immune infiltration. Immunohistochemistry (IHC) method was used to detect the expression level of RDM1 in 90 cases of hepatocellular carcinoma and adjacent normal liver tissues.Results: RDM1 expression was up-regulated in most cancers. The expression of RDM1 was remarkably higher than that of the corresponding normal control genes in LIHC tissues. The increase in RDM1 messenger RNA (mRNA) expression was closely related to the decreases in overall survival (OS) and progression-free survival (PFS). Additionally, the increase in RDM1 mRNA expression was closely related to the infiltration levels of macrophages, CD8+ T cells and B cells and was positively correlated with a variety of immune markers in LIHC.Conclusion: The findings of the present study demonstrate that RDM1 is a potentially valuable prognostic biomarker that can help determine the progression of cancer and is associated with immune cell infiltration in LIHC.

The role of different PI3K protein subtypes in the metastasis, angiogenesis and clinical prognosis of hepatocellular carcinoma

Background and objectivesAbnormal activation of the PI3K/AKT pathway is closely related to tumor occurrence, development and angiogenesis. PI3K, as a key protein in the PI3K/Akt pathway, has different subtypes that play diverse roles in various tumors. The aim of this study was to examine the roles of different PI3K protein subtypes (PI3Kp110α, PI3Kp110β, and PI3Kp110δ) in the metastasis, angiogenesis and prognosis of hepatocellular carcinoma (HCC). MethodsThe roles of different PI3K protein subtypes in the metastasis, angiogenesis and prognosis of HCC were assessed by immunohistochemical staining of 97 HCC tissues and the STRING database. ResultsOur results showed that PI3Kp110α and PI3Kp110δ were associated with HCC metastasis and angiogenesis. Patients with high expression of PI3Kp110α and PI3Kp110δ had a worse prognosis and shorter survival time, respectively, than those with low expression, whereas these effects were not observed for PI3Kp110β. Cox regression analysis showed that PI3Kp110α and clinical stage were independent risk factors for the overall survival of HCC patients. ConclusionsPI3Kp110α and PI3Kp110δ promoted HCC metastasis and angiogenesis via the PI3K/AKT pathway, and PI3Kp110α was an independent risk factor for HCC patients. These findings provide valuable insights for the prognosis evaluation and the selection of subtype inhibitors of HCC patients.

Effects of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α overexpression on hepatocellular carcinoma survival: A systematic review with meta-analysis.

The role of hypoxia-inducible factor-1α (HIF-1α) and hypoxia-inducible factor-2α (HIF-2α) has been implicated in the clinical prognosis of hepatocellular carcinoma (HCC), but the results remain controversial. We aim to investigate the association of HIF-1α and HIF-2α overexpression with the prognosis and clinicopathological features of HCC. A systematic search was conducted in PubMed, Embase, Scopus, Web of Science, and Cochrane Library until June 20, 2020. Meta-analysis was conducted to generate combined HRs with 95% confidence intervals (CI) for overall survival (OS) and disease-free survival (DFS). Odds ratios (ORs) with 95% CI were also derived by fixed or random effect model. Twenty-two studies involving 3238 patients were included. Combined data suggested that overexpression of HIF-1α in HCC was not only correlated with poorer OS [HR=1.75 (95% CI: 1.53-2.00)] and DFS [HR=1.64 (95% CI: 1.34-2.00)] but was also positively associated with vascular invasion [OR=1.83 (95% CI: 1.36-2.48)], tumor size [OR=1.36 (95% CI: 1.12-1.66)], and tumor number [1.74 (95% CI: 1.34-2.25)]. In contrast, HIF-2α overexpression was not associated with the prognosis and clinicopathological features of HCC. Our data provided compelling evidence of a worse prognosis of HCC in HIF-1α overexpression patients but not HIF-2α overexpression ones.

The Effect of m6A Methylation Regulatory Factors on the Malignant Progression and Clinical Prognosis of Hepatocellular Carcinoma.

The modification level of the transcript N6-methyladenosine (m6A), dynamically regulated by methyltransferases, binding proteins and demethylases, is closely related to the occurrence, and progression of tumors. Here, 13 differentially expressed m6A methylation regulators were confirmed in 374 hepatocellular carcinoma (HCC) patients, among which RBM15, YTHDC1, YTHDF1, and YTHDF2 were significantly variant in different stages and grades. Further consensus clustering analysis identified two HCC subtypes (cluster1/2) in this cohort, finding an active role of the m6A methylation regulators in the malignant progression of HCC. Furthermore, GESA enrichment analysis showed that PPAR signaling pathway, and the pathways involved in retinol metabolism and peroxisome were related to tumor progression. Additionally, a 4-gene risk model (ROC = 0.729) that can be used as a prognostic marker and a predictor for clinicopathological characteristics of HCC was constructed via univariate and multivariate Cox regression analyses. Analysis on overall survival and disease-free survival demonstrated that METTL3 and YTHDF1 out of the four genes in the model could serve as independent prognostic factors for HCC. Overall, this study systematically investigated the effect of m6A methylation regulators on the malignant progression of HCC and proposed a 4-gene risk prediction model, laying a theoretical foundation for the further research on HCC prognosis.

N6-methyladenosine (m6A) RNA methylation regulators are associated with clinical prognosis in hepatocellular carcinoma.

BackgroundN6-methyladenosine (m6A) methylation is a common class of RNA modification. Similar to DNA methylation, m6A methylation regulates most mRNA expressions. At present, most research has found that m6A methylation is related to tumorigenesis and development; however, there are few studies about hepatocellular carcinoma (HCC). This study aimed to analyze the expression level of m6A methylation regulators and their correlation on the clinical features in HCC.MethodsA total of 13 m6A methylation regulators were evaluated. mRNA data and clinical information were obtained from the Cancer Genome Atlas (TCGA). The Wilcoxon test was utilized to analyze the differences between m6A RNA methylation regulators, and Pearson’s test was used to test the correlation between them. We constructed a tumor subgroup model based on the 13 molecules used for the analysis of the correlations with the clinical features. Two genes (ZC3H13 and YTHDF2) screened by Cox and LASSO regression were used to construct a tumor risk model for analyzing the correlations with clinical features. Finally, we verified the expression of the two molecules in liver cancer and adjacent tissues by Western blot and real-time polymerase chain reaction (PCR) (n=6). P<0.05 was considered statistically significant.ResultsEleven of the 13 molecules were higher in the liver cancer tissues than the adjacent tissues (P<0.05), and most were significantly positively related. Two subgroup models were constructed. Subgroup 2 patients had higher levels of alpha-fetoprotein (AFP), while grade and the three-year survival were lower than subgroup 1 (49% vs. 77%) with significant differences (P<0.05). The risk model suggested that patients in the high-risk group showed high AFP levels, and the 3- and 5-year survival rates were lower than the low-risk group (3-year survival rate: 19% vs. 31%, 5-year survival rate: 12% vs. 17%). The Western blot test showed that the expression of YTHDF2 in the liver cancer tissues was greater than that in the precancerous tissues (P<0.05), while the expression of ZC3H13 was not significant. Real-time PCR showed that the expression of YTHDF2 mRNA in liver cancer tissues were higher than that in adjacent tissues (7.64±0.44 vs. 4.99±0.61, P=0.006), while the expression of ZC3H13 mRNA had no statistical difference (5.56±0.18 vs. 5.42±0.33, P>0.05). The results of the in vitro experiment were consistent with bioinformatic analysis.ConclusionsThe abnormal expression of m6A methylation regulators in the liver tissues suggest that m6A may play an important role in the development of HCC. Tumor models we constructed that could effectively predict the prognosis of patients, and the clinical correlation results were consistent with clinical practices. Our research is expected to provide a reference for the prognostic stratification and treatment strategy development of HCC.

The Cancer Genome Atlas (TCGA) based m6A methylation-related genes predict prognosis in hepatocellular carcinoma

ABSTRACT The current study aims to investigate the significance of N 6-methyladenosine (m6A) methylation-related genes in the clinical prognosis of hepatocellular carcinoma (HCC) using bioinformatics analyses based on The Cancer Genome Atlas (TCGA) database. Transcriptome data and corresponding clinical data on m6A methylation-related genes (including 15 genes) were obtained from TCGA database. Differential expression of 15 genes was identified. Survival curves of subgroups based on m6A methylation-related gene expression levels were plotted. We selected potential predictive genes and analyzed their prognostic values using bioinformatics methods. Eleven genes (METTL3, YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, FTO, KIAA1429, HNRNPC, HNRNPA2B1, and RBM15) were found to be overexpressed in HCC. Of these, five genes had worse survival (P < 0.05). There was a significant difference in the survival rate between subgroups with different expression levels of m6A. We selected five potential predictors (METTL3, KIAA1429, ZC3H13, YTHDF1, and YTHDF2) that met the independent predictive value. ZC3H13 was upregulated in patients with high cancer risk, whereas METTL3, KIAA1429, YTHDF1, and YTHDF2 were downregulated. In summary, we found that the expression levels of m6A methylation-related genes were different in patients with HCC and correlated with survival and prognosis. This implies that m6A methylation-related genes may be promising prognostic indicators or therapeutic targets for HCC.

Piperlongumine Achieves Antitumor Effects by Targeting Angiogenic Genes that Have Predictive Value for the Diagnosis, Prognosis and Recurrence of Hepatocellular Carcinoma

Background: In the context of the high recurrence rate and poor clinical prognosis of hepatocellular carcinoma (HCC), and considering the indispensable effect of angiogenesis in the development and metastasis of tumors, we analyzed HCC-related RNA sequence data to identify angiogenic genes closely associated with the prognosis of HCC. We used the identified genes to establish diagnostic, prognostic, and recurrence models to help clinicians effectively develop individualized treatment strategies for HCC. Method: We comprehensively analyzed 132 angiogenic genes and HCC-related RNA sequence data from the TCGA and the ICGC databases by Cox regression and least absolute shrinkage and selection operator(LASSO) regression to identify angiogenic genes; the genes were used to establish prognosis, recurrence and diagnostic models and to construct corresponding predictive nomograms. Pharmacological analysis was performed to detect target angiogenic drugs. Subsequently, in vitro cell experiments and western blot experiments were used to explore the anti-tumor effect and possible molecular mechanism of an angiogenesis-targeting target drug. Results: Four angiogenic genes (ENFA3, EGF, MMP3 and AURKB) were used to establish prognostic, relapse and diagnostic models. Through the prognostic model, we found that the overall survival (OS) of the high-risk group in the training cohort (P < 0.001, HR = 0.33) and validation cohort (P < 0.001, HR = 0.33) were significantly worse than those of the low-risk group. The recurrence model also demonstrated that in the training cohort (P<0.001, HR= 0.42) and validation cohort (P=0.01, HR=0.5), the recurrence rate of the high-risk group was significantly higher than that of the low-risk group. In addition, angiogenic genes were an independent predictor of prognosis and recurrence (P < 0.05). The diagnostic model has been validated in terms of correctly distinguishing between HCC and normal samples, as well as between HCC and proliferative nodule samples, with high sensitivity and high specificity. Through pharmacological analysis, we identified piperlongumine as a drug for targeting angiogenesis. Subsequent targeted drug cell experiments suggested that the anti-tumor effect of piperlongumine was achieved by inhibiting angiogenesis via the EGF/EGFR axis. Conclusion: We identified and validated the predictive properties of the diagnostic, recurrence, and prognostic models of four key angiogenic genes and confirmed the value of these four genes for constructing models. Piperlongumine was confirmed to inhibit HCC cell proliferation and angiogenesis via the EGF/EGFR axis. These findings may help to provide new individualized treatment ideas for HCC patients. Funding Statement: This study was supported by National Natural Science Foundation of China (Nos. 81803778), and The Key Research and development Project of Zhejiang Province (No. 2018C0302), and Public Welfare Project of Zhejiang Province (Nos. LGF19H180010, LGD19H160002 and LGF19H180009), and Medical and Health Care Key Project of Zhejiang Province (Nos. 2016146810, 2018KY197 and 2018KY932). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Since these mRNA-sequencing data were obtained from TCGA and ICGC databases and were freely available to the public, and as this study also strictly followed the publication guidelines and access policies of these databases, this study does not require ethical review and approval from an Ethics Committee.

IL-10 promoter hypomethylation is associated with increased IL-10 expression and poor survival in hepatocellular carcinoma.

BackgroundEpigenetic alterations of tumor-associated genes contribute to the pathogenesis of virtually all cancer types. We evaluated the methylation status of the interleukin-10 (IL-10) gene promoter and assessed its association with IL-10 mRNA expression and clinical prognosis in hepatocellular carcinoma (HCC) patients.MethodsMethylation-specific polymerase chain reaction (MSP) and real-time polymerase chain reaction (PCR) were used to define the methylation index (MI) of the IL-10 gene and quantify IL-10 mRNA expression in 120 HCC samples and paired non-tumor tissues.ResultsMean MI was 0.47 in HCC specimens and 0.59 in non-tumor controls, and was associated with metastasis classification and serum α-fetoprotein (AFP) levels. IL-10 mRNA levels [mean –∆∆Ct of 1.678 in HCC cases with hypomethylation (∆MI ≤0) and –0.18 in HCC cases with hypermethylation (∆MI >0)] also correlated with metastasis classification and serum AFP. An association was detected between IL-10 mRNA and its gene’s MI in HCC. Also, an association was found between IL-10 hypomethylation, but not IL-10 mRNA expression and reduced postoperative HCC survival.ConclusionsThese results indicate that IL-10 promoter hypomethylation is associated with increased IL-10 mRNA levels and indicative of poor survival in HCC.

Inhibition of Polo-Like Kinase 1 by BI2536 Reverses the Multidrug Resistance of Human Hepatoma Cells In Vitro and In Vivo.

Multi Drug Resistance (MDR) is the phenomenon that cancers develop resistance to majority of chemotherapy drugs and is a serious obstacle to the treatment for Hepatocellular Carcinoma (HCC). Polo-Like Kinase 1 (PLK1) is a serine/threonine kinase associated with tumor growth and clinical prognosis in HCC and BI2536 is its potent inhibitor with IC50 of 0.83nM. To test whether the down-regulation of PLK1 by its inhibitor BI2536 would have beneficial effects on the reversal of MDR in HCC cells. The CCK-8 assay was used to determine the viability of HepG2/ADM and SMMC7721/ADM cells and their parental cells treated with BI2536. Then animal model studies were performed. Cell invasion assay and wound healing assay were used to determine the invasion ability and motility. Flow cytometric was used to test the apoptosis induced by BI2536. Western blot and quantitative real-time PCR were performed to test the change of expression of MDR and apoptosis-related gene. BI2536 down-regulated the expression of PLK1 protein and mRNA specifically. BI2536 can significantly reduce IC50 for ADM and other drugs in ADM-resistant HCC cells. Meanwhile, it inhibited cell viability, proliferation, and invasion, and induced cell cycle arrest and apoptosis in HCC cells with MDR. Our results suggest that PLK1 inhibitor BI2536 can re-sensitize HCC cancer cell with MDR through induction of apoptosis. Thus, PLK1 inhibitor BI2536 may act as an effective chemotherapeutic drug in the clinical treatment of HCC patients with MDR.

The long noncoding RNA AK002107 negatively modulates miR-140-5p and targets TGFBR1 to induce epithelial-mesenchymal transition in hepatocellular carcinoma.

The abnormal expression of long noncoding RNAs (lncRNAs) is associated with human carcinoma. The present study aimed to investigate the mechanisms underlying the function of lncRNA AK002107 in the progression of hepatocellular carcinoma (HCC). The differential expression of lncRNAs between HCC and paired nontumor tissues was identified using microarrays, and the correlation between the expression of lncRNA AK002107 and the clinical prognosis of HCC was analyzed. We investigated the role of lncRNA AK002107 in HCC tumor biology in vitro using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT), colony formation, and Matrigel invasion assays and in vivo by assessing the growth of xenografted HCC tumors. The potential microRNAs that interact with lncRNA AK002107 were identified using online tools and were verified using PCR and luciferase reporter assay. The levels of TGFBR1, E‐cadherin, and vimentin were determined using western blot assays. We then further investigated the correlation between expression of lncRNA AK002107 with miR‐140‐5p and TGFBR1 expression in HCC tissues. The expression of lncRNA AK002107 is frequently upregulated in HCC samples and cell lines. Patients with HCC who have elevated lncRNA AK002107 expression exhibit poorer overall survival and disease‐free survival. Silencing lncRNA AK002107 expression significantly inhibited HCC cell proliferation, colony formation, and invasion both in vitro and in vivo. Furthermore, lncRNA AK002107 directly binds to miR‐140‐5p and significantly inhibits miR‐140‐5p expression. The functions of lncRNA AK002107 in cell growth and tumor invasion are mediated via miR‐140‐5p. lncRNA AK002107 upregulated TGFBR1 expression and then induced epithelial–mesenchymal transition (EMT) by inhibiting miR‐140‐5p expression. The expression of lncRNA AK002107 inversely correlated with miR‐140‐5p expression and positively correlated with TGFBR1 expression in HCC tissues. In summary, lncRNA AK002107 functions as an oncogene in tumors by inhibiting miR‐140‐5p, targeting TGFBR1, and then inducing EMT. The lncRNA AK002107/miR‐140‐5p/TGFBR1/EMT regulatory network may be a valuable target for the development of novel diagnostic and treatment methods for HCC.

Overexpression of Acyl-CoA Ligase 4 (ACSL4) in Patients with Hepatocellular Carcinoma and its Prognosis.

BackgroundRecently, accumulating studies have found that ACSL4 dysregulation is related to a great number of malignant tumors. The purpose of the present study was to explore the relationship between ACSL4 expression level and clinical prognosis of hepatocellular carcinoma (HCC) patients.Material/MethodsThe Oncomine and TCGA databases were used to predict the expression of ACSL4 mRNA in HCC and its association with HCC prognosis. Further, immunohistochemistry was performed to verify the ACSL4 protein expression in 116 paired HCC and adjacent normal tissues. Kaplan-Meier and cox analysis were performed to validate the correlation between ACSL4 expression and HCC prognosis.ResultsWe first used the Oncomine database to find that ACSL4 mRNA expression level was significantly higher in HCC tissues than that in normal tissues (p all <0.001). The results were consistent with those in the TCGA database. Then, immunohistochemical results demonstrated that the ACSL4 positive expression rate was 70.7% in HCC tissues. ACSL4 differential expression level was significantly related to Edmondson grade (p=0.010), AFP (p=0.001) and TNM stage (p=0.012). Survival analysis revealed that both overall survival (OS) and disease-free survival (DFS) time were remarkably reduced in HCC patients with ACSL4 high expression (p=0.001 and 0.000, respectively). Moreover, Cox multivariate analysis demonstrated that ACSL4 expression was the only independent prognostic factor for both OS and DFS (both p values=0.001).ConclusionsTaken together, our study demonstrated that ACSL4 was overexpressed in HCC, and it will be a new potential therapeutic target for HCC as an independent adverse prognostic parameter.

High expression of miR-105-1 positively correlates with clinical prognosis of hepatocellular carcinoma by targeting oncogene NCOA1

Increasing evidence supports that microRNA (miRNA) plays a significant functional role in cancer progression by directly regulating respective targets. In this study, the expression levels of miR-105-1 and its target gene were analyzed using genes microarray and hierarchical clustering analysis followed by validation with quantitative RT-PCR in hepatocellular carcinoma (HCC) and normal liver tissues. We examined the expression of nuclear receptor coactivator 1 (NCOA1), the potential target gene of miR-105-1, following the transfection of miR-105-1 mimics or inhibitors. Our results showed that miR-105-1 was downregulated in HCC tissues when compared with normal liver tissues and patients with lower miR-105-1 expression had shorter overall survival (OS) and progression free survival (PFS). Moreover, NCOA1 was confirmed to be a direct target of miR-105-1. Furthermore, concomitant high expression of NCOA1 and low expression of miR-105-1 correlated with a shorter median OS and PFS in HCC patients. In conclusion, our results provide the first evidence that NCOA1 is a direct target of miR-105-1 suggesting that NCOA1 and miR-105-1 may have potential prognostic value and may be useful as tumor biomarkers for the diagnosis of HCC patients.

Genomic Aberrations in the HTPAP Promoter Affect Tumor Metastasis and Clinical Prognosis of Hepatocellular Carcinoma

We previously reported that the intronic tagSNP +357G/C in the metastasis suppressor HTPAP is associated with metastasis and prognosis of hepatocellular carcinoma (HCC). The aim of this study was to investigate whether SNPs in the HTPAP promoter modulate HTPAP expression and prognosis of HCC. Genomic DNA from 572 microdissected HCCs were genotyped by pyrosequencing and verified by direct sequencing. Haplotype blocks were analyzed. Reporter plasmids were constructed and transfected into HCC cell lines. Transcriptional activities of plasmids were analyzed by dual-luciferase reporter systems. HTPAP expression was measured by real-time quantitative PCR, western blots, and tissue microarrays. Invasion was assessed by Matrigel assays. The prognostic values of HTPAP promoter SNPs in HCC were evaluated by Kaplan-Meier and Cox regression analyses. We identified six SNPs, including -1053A/G and +64G/C, in the HTPAP promoter. The SNPs were in complete linkage disequilibrium, resulting in three promoter haplotypes (promoter I:-1053AA/+64GG, promoter II: -1053AG/+64GC, and promoter III: -1053GG/+64CC). Promoter I manifested the highest luciferase index (p<0.005). However, no significant difference was observed between promoters II and III. We consistently found that HTPAP mRNA and protein levels were significantly higher in promoter I than that of promoter II+III (p<0.001). Invasion was increased in HCC cells transfected with promoters II+III compared to those transfected with promoter I (p<0.05). The HTPAP promoter II+III haplotype was associated with significantly increased metastasis compared to that of promoter I (p = 0.023). The postoperative five-year overall survival of patients with promoters II+III was lower than that of patients with promoter I (p = 0.006). Multivariate analysis showed that the promoter II+III haplotype was an adverse prognostic marker in HCC. The genetic variants at loci –1053 and +64 of the HTPAP promoter affect the expression of HTPAP, which might be a novel determinant and target for HCC prognosis.