Adjacent Non-tumor Liver Tissues Research Articles

Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has a potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

Characterization of tumor microbiome and associations with prognosis in intrahepatic cholangiocarcinoma.

The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.

Evaluation of potential targets to enhance the sensitivity of cholangiocarcinoma cells to anticancer drugs

BackgroundCholangiocarcinoma (CCA) is a highly lethal cancer originated in the biliary tree. Available treatments for CCA are scarcely effective, partly due to mechanisms of chemoresistance, such as aberrant activation of Wnt/β-catenin pathway and dysfunctional p53. AimTo evaluate the impact of enhancing the expression of negative regulators of the Wnt/β-catenin pathway (AXIN1, AXIN2, and GSK3B) and the tumor suppressor gene TP53. MethodsGene expression in paired samples of CCA and adjacent non-tumor liver tissue was determined by RT-qPCR and immunohistochemistry (IHC). Using lentiviral vectors, CCA cells were transduced with genes of interest to assess their impact on the resistome (TLDA), apoptosis (annexin V/propidium iodide), and decreased cell viability (MTT). ResultsIHC revealed marked nuclear localization of β-catenin, consistent with Wnt/β-catenin pathway activation. In silico analysis with data from TCGA showed heterogeneous down-regulation of AXIN1, AXIN2, and GSK3B in CCA. Enhancing the expression of AXIN1, AXIN2, and GSK3B in CCA cells was not enough to block the activity of this signaling pathway or significantly modify resistance to 5-FU, gemcitabine, and platinated drugs. Consistent with impaired p53 function, CDKN1A was down-regulated in CCA. Forced TP53 expression induced p21 up-regulation and reduced cell proliferation. Moreover, the resistome was modified (FAS, BAX, TYMP, and CES2 up-regulation along with DHFR, RRM1, and BIRC5 down-regulation), which was accompanied by enhanced sensitivity to some antitumor drugs, mainly platinated drugs. ConclusionEnhancing TP53 expression, but not that of AXIN1, AXIN2, and GSK3B, in CCA cells may be a useful strategy to sensitize CCA to antitumor drugs.

EZH2-mediated epigenetic silencing of tumor-suppressive let-7c/miR-99a cluster by hepatitis B virus X antigen enhances hepatocellular carcinoma progression and metastasis

BackgroundHepatitis B virus (HBV)-encoded X antigen, HBx, assists in the development of hepatocellular carcinoma (HCC) through complex mechanisms. Our results provide new insights into the EZH2 epigenetic repression of let-7c that promotes HCC migration induced by HBx. Thus, let-7c and HMGA2 represent key diagnostic markers and potential therapeutic targets for the treatment of HBV-related HCC.ResultsWe investigated the epigenetic regulation of let-7c, an important representative miRNA in liver tumor metastasis, in human HCC cells to verify the effect of HBx. Based on quantitative PCR (qPCR) of mRNA isolated from tumor and adjacent non-tumor liver tissues of 24 patients with HBV-related HCC, EZH2 expression was significantly overexpressed in most HCC tissues (87.5%). We executed a miRNA microarray analysis in paired HBV-related HCC tumor and adjacent non-tumorous liver tissue from six of these patients and identified let-7c, miR-199a-3p, and miR-99a as being downregulated in the tumor tissue. Real-time PCR analysis verified significant downregulation of let-7c and miR-99a in both HepG2X and Hep3BX cells, which stably overexpress HBx, relative to parental cells. HBX enhanced EZH2 expression and attenuated let-7c expression to induce HMGA2 expression in the HCC cells. Knockdown of HMGA2 significantly downregulated the metastatic potential of HCC cells induced by HBx.ConclusionsThe deregulation of let-7c expression by HBx may indicate a potential novel pathway through deregulating cell metastasis and imply that HMGA2 might be used as a new prognostic marker and/or as an effective therapeutic target for HCC.

Tumor-associated endothelial cell prognostic risk model and tumor immune environment modulation in liver cancer based on single-cell and bulk RNA sequencing: Experimental verification

BackgroundTo build a prognostic and immunotherapeutic response prediction model for liver cancer based on marker genes of tumor-associated endothelial cell (TEC). MethodSingle cell sequencing data from Gene Expression Omnibus (GEO) liver cancer patients were utilized to identify TEC subpopulations. Models were built from transcriptomic and clinical data of TCGA liver cancer patients. The GSE76427 and ICGC databases were used as independent validation sets. Time-dependent receiver operating characteristic (ROC) curves and Kaplan-Meier curves were used to verify the ability of the model to predict survival. XCELL, TIMER, QUANTISEQ, CIBERSORT, CIBERSORT-ABS, and ssGSEA were applied to evaluate tumor immune cell infiltration. The TIDE score was used to predict the effect of immunotherapy. Immune blockade checkpoint gene, tumor mutational load and GSVA enrichment analyses were further explored. The expression levels of candidate genes were measured and validated by real-time PCR between liver cancer tissues and adjacent nontumor liver tissues. ResultsEighty-seven genes were identified as marker genes for TECs. IGFBP3, RHOC, S100A16, FSCN1, and CLEC3B were included in the constructed prognostic model. Time-dependent ROC curve values were higher than 0.700 in both the model and validation groups. The low risk group exhibited high immune cell infiltration and function than the higher risk group. The TIDE score indicated that the low-risk group benefited more from immunotherapy than the high-risk group. The risk score and multiple immune blockade checkpoint genes and immune-related pathways were strongly correlated. ConclusionNovel signatures of TEC marker genes showed a powerful ability to predict prognosis and immunotherapy response in patients with liver cancer.

Extrachromosomal circular MiR-17-92 amplicon promotes HCC.

Extrachromosomal circular DNAs (eccDNAs) are prevalent in cancer genomes and emerge as a class of crucial yet less characterized oncogenic drivers. However, the structure, composition, genome-wide frequency, and contribution of eccDNAs in HCC, one of the most fatal and prevalent cancers, remain unexplored. In this study, we provide a comprehensive characterization of eccDNAs in human HCC and demonstrate an oncogenic role of microRNA (miRNA)-17-92-containing eccDNAs in tumor progression. Using the circle-sequencing method, we identify and characterize more than 230,000 eccDNAs from 4 paired samples of HCC tumor and adjacent nontumor liver tissues. EccDNAs are highly enriched in HCC tumors, preferentially originate from certain chromosomal hotspots, and are correlated with differential gene expression. Particularly, a series of eccDNAs carrying the miRNA-17-92 cluster are validated by outward PCR and Sanger sequencing. Quantitative PCR analyses reveal that miRNA-17-92-containing eccDNAs, along with the expression of their corresponding miRNAs, are elevated in HCC tumors and associated with poor outcomes and the age of HCC patients. More intriguingly, exogenous expression of artificial DNA circles harboring the miR-17-92 cluster, which is synthesized by the ligase-assisted minicircle accumulation method, can significantly accelerate HCC cell proliferation and migration. These findings delineate the genome-wide eccDNAs profiling of HCC and highlight the functional significance of miRNA-containing eccDNAs in tumorigenesis, providing insight into HCC pathogenesis and cancer therapy, as well as eccDNA and miRNA biology.

SAA1 identified as a potential prediction biomarker for metastasis of hepatocellular carcinoma via multi-omics approaches.

Metastasis is the major cause of high recurrence and mortality of hepatocellular carcinoma (HCC). Unfortunately, there are few reports on effective biomarkers of HCC metastasis. Previous studies have reported that SAA1 may be a predictor and prognostic biomarker for multiple malignant tumors. However, the role of SAA1 in HCC has not yet been investigated. We applied RNA sequencing and proteomics analysis to investigate the expression landscape of HCC cell lines and patient serum, respectively. SAA1 is a common key gene and listed as a candidate biomarker of HCC metastasis. It was validated in two cell lines, 107 participants serum, and 63 matched HCC and adjacent non-tumorous liver tissues. Human Protein Atlas (HPA), Genotype-Tissue Expression (GTEx), and The Cancer Genome Atlas (TCGA) datasets were integrated to explore SAA1 expression among various cell types and organs. The diagnostic and prognostic value of SAA1 in HCC were determined through receiver operating characteristic (ROC) and Kaplan-Meier curves. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction (PPI) network were constructed for SAA1, as well as for its co-expressed genes. We further analyzed the correlation between SAA1 and co-expression genes. We found 7 differentially expressed genes (DEGs) and 14 differentially expressed proteins (DEPs) were related to HCC metastasis. SAA1, a key candidate biomarker, was highly enriched in hepatocytes and liver organ, and it was also highly expressed in HCC cells and the serum and tissues of HCC patients. The results of ROC curve analysis indicated that SAA1 had better predictive values for distinguishing HCC metastasis from non-metastasis. Kaplan-Meier curve analysis revealed that HCC patients with higher SAA1 expression had worse overall survival. Our findings provide new insights into HCC metastasis by identifying candidate gene prediction biomarkers for HCC metastasis.

Clinical Significance of Combined Epithelial-Mesenchymal Transition Markers Expression and Role of Rac1 in Hepatocellular Carcinoma.

Epithelial-mesenchymal transition (EMT) has been implicated in cancer progression, invasion, and metastasis. We aimed to evaluate the correlations between clinicopathological characteristics and EMT markers in patients with hepatocellular carcinoma (HCC) who underwent surgical resection and to identify the key regulator in EMT process. Fresh-frozen HCC tissues and adjacent nontumor liver tissues from 30 patients who underwent surgical resection were provided by the Gachon University Gil Medical Center Bio Bank. Human HCC cell lines, Hep3B, SNU449, and Huh7 cells were transfected with Rac1 siRNA and exposed to hypoxic conditions. The combined EMT markers expression (down-expression of E-cadherin and overexpression of p21-activated kinases 1 (PAK1)/Snail) by Western blot in HCC tissues when compared to adjacent nontumor liver tissues was significantly associated with macrovascular invasion (p = 0.021), microvascular invasion (p = 0.001), large tumor size (p = 0.021), and advanced tumor stage (p = 0.015). Patients with combined EMT markers expression showed early recurrence and poor overall survival. In vitro studies showed that Rac1 knockdown decreased the expression of EMT markers including PAK1 and Snail in hypoxia-induced Hep3B cells and suppressed the migration and invasion of hypoxia-induced HCC cells. Rac1 may be a potential therapeutic target for inhibition of EMT process through the inhibition of PAK1 and Snail in HCC.

Clinical and prognostic significance of CCPG1 in hepatocellular carcinoma.

This study aimed to examine the clinical and prognostic significance of cell-cycle progression gene 1 (CCPG1) in hepatocellular carcinoma (HCC). We firstly analyzed CCPG1 expression in various cancers using The Cancer Genome Atlas and the Genotype-Tissue Expression project databases. The relative expression levels of CCPG1 were determined in 164 paired HCC and adjacent tissues using immunohistochemistry. The correlation between CCPG1 and clinicopathological characteristics of HCC was analyzed. Cox proportional models were used to identify the prognostic factors for overall survival (OS) and disease-free survival (DFS). The expression of CCPG1 was lower in HCC tissues than in adjacent non-tumor liver tissues. The expression of CCPG1 was significantly correlated with tumor number (p = 0.02) and tumor differentiation (p = 0.04) in HCC. Lower expression of CCPG1 in HCC patients was associated with poor OS and DFS (p < 0.01). Relative low expression of CCPG1 in HCC is significantly correlated with the poor prognosis of HCC patients after surgical resection, suggesting its possible role as a potential prognostic marker for HCC.

Effect of miRNA-200b on the proliferation of liver cancer cells via targeting SMYD2/p53 signaling pathway.

Our previous study has verified that high level of SET and MYND domain-containing protein 2 (SMYD2) plays an important role in acquiring aggressive ability for liver cancer cells in hepatocellular carcinoma. MiR-200b as a tumor suppressor gene involves in a variety of cancers. This study aims to investigate the correlation between miR-200b and SMYD2 in hepatocellular carcinoma and the underlying mechanism. Firstly, the levels of SMYD2 and miR-200b in hepatocellular carcinoma tissues and matched adjacent non-tumor liver tissues were tested with real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Secondly, we evaluated the interaction between miR-200b and SMYD2 using dual-luciferase reporter assay. Thirdly, we elucidated the effect of miR-200b on SMYD2 and its downstream targets p53/CyclinE1. Finally, we silenced SMYD2 in hepatocellular carcinoma cell lines to investigate its effect on tumor proliferation and cell cycle progression, and further confirmed the correlation among SMYD2 and p53/CyclinE1. Compared with the matched adjacent non-tumor liver tissues, miR-200b was obviously decreased, and SMYD2 was significantly increased in hepatocellular carcinoma (both P<0.05). Spearman's rank correlation revealed that miR-200b expression was negatively correlated with SMYD2 (P<0.01). Computer algorithm and dual-luciferase reporter assay revealed that miR-200b directly targeted and suppressed SMYD2 in HEK 293T cells. The down-regulated miR-200b expression promoted hepatoma cell proliferation (P<0.05) and increased SMYD2 expression(P<0.01), while the up-regulated expression of miR-200b had an opposite effect. The knockdown of SMYD2 suppressed the proliferation of MHCC-97L cells (P<0.01), down-regulated CyclinE1, and up-regulated p53 expression (both P<0.05). MiR-200b is involved in hepatocellular carcinoma progression via targeting SMYD2 and regulating SMYD2/p53/CyclinE1 signaling pathway and may be used as a potential target for hepatocellular carcinoma treatment.

Analysis of viral integration reveals new insights of oncogenic mechanism in HBV-infected intrahepatic cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma

BackgroundIntegration of HBV DNA into the human genome could progressively contribute to hepatocarcinogenesis. Both intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC) are known to be associated with HBV infection. However, the integration of HBV and mechanism of HBV-induced carcinogenesis in ICC and CHC remains unclear.Methods41 patients with ICC and 20 patients with CHC were recruited in the study. We conducted HIVID analysis on these 61 samples to identify HBV integration sites in both the tumor tissues and adjacent non-tumor liver tissues. To further explore the effect of HBV integration on gene alteration, we selected paired tumors and adjacent non-tumor liver tissues from 3 ICC and 4 CHC patients for RNA-seq and WGS.ResultsWe detected 493 HBV integration sites in ICC patients, of which 417 were from tumor samples and 76 were from non-tumor samples. And 246 HBV integration sites were detected in CHC patients, of which 156 were located in the genome of tumor samples and 90 were in non-tumor samples. Recurrent HBV integration events were detected in ICC including TERT, ZMAT4, MET, ANKFN1, PLXNB2, and in CHC like TERT, ALKBH5. Together with our established data of HBV-infected hepatocellular carcinoma, we found that HBV preferentially integrates into the specific regions which may affect the gene expression and regulation in cells and involved in carcinogenesis. We further performed genomic and transcriptomic sequencing of three ICC and four CHC patients, and found that HBV fragments could integrate near some important oncogene like TERT, causing large-scale genome variations on nearby genomic sequences, and at the same time changing the expression level of the oncogenes.ConclusionComparative analysis demonstrates numerous newly discovered mutational events in ICC and CHC resulting from HBV insertions in the host genome. Our study provides an in-depth biological and clinical insights into HBV-induced ICC and CHC.

CDC20 May Serve as a Potential Biomarker-Based Risk Score System in Predicting the Prognosis of Patients with Hepatocellular Carcinoma.

Background The specificity and sensitivity of hepatocellular carcinoma (HCC) diagnostic markers are limited, hindering the early diagnosis and treatment of HCC patients. Therefore, improving prognostic biomarkers for patients with HCC is urgently needed. Methods HCC-related datasets were downloaded from the public databases. Differentially expressed genes (DEGs) between HCC and adjacent nontumor liver tissues were then identified. Moreover, the intersection of DEGs in four datasets (GSE138178, GSE77509, GSE84006, and TCGA) was used in the functional enrichment, and module genes were obtained by a coexpression network. Cox and Kaplan-Meier analyses were used to identify overall survival- (OS-) related genes from module genes. Area under the curve (AUC) > 0.9 of OS-related genes was then carried out in order to perform the protein-protein interaction network. The feature genes were identified by least absolute shrinkage and selection operator (LASSO). Furthermore, the hub gene was identified through the univariate Cox model, after which the correlation analysis between the hub gene and pathways was explored. Finally, infiltration in immune cell types in HCC was analyzed. Results A total of 2,227 upregulated genes and 1,501 downregulated DEGs were obtained in all four datasets, which were mainly found to be involved in the cell cycle and retinol metabolism. Accordingly, 998 OS-related genes were screened to construct the LASSO model. Finally, 8 feature genes (BUB1, CCNB1, CCNB2, CCNA2, AURKB, CDC20, OIP5, and TTK) were obtained. CDC20 was shown to serve as a poor prognostic gene in HCC and was mainly involved in the cell cycle. Moreover, a positive correlation was noted between the high degree of infiltration with Th2 and CDC20. Conclusion High expression of CDC20 predicted poor survival, as potential target in the treatment for HCC.

Distinctive Metabolism-Associated Gene Clusters That Are Also Prognostic in Intrahepatic Cholangiocarcinoma and Hepatocellular Carcinoma.

Objective To offer new prognostic evaluations by exploring potentially distinctive genetic features of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Methods There were 12 samples for gene expression profiling processes in this study. These included three HCC lesion samples and their matched adjacent nontumor liver tissues obtained from patients with HCC, as well as three ICC samples and their controls collected similarly. In addition to the expression matrix generated on our own, profiles of other cohorts from The Cancer Genome Atlas (TCGA) program and the Gene Expression Omnibus (GEO) were also employed in later bioinformatical analyses. Differential analyses, functional analyses, protein interaction network analyses, and gene set variation analyses were used to identify key genes. To establish the prognostic models, univariate/multivariate Cox analyses and subsequent stepwise regression were applied, with the Akaike information criterion evaluating the goodness of fitness. Results The top three pathways enriched in HCC were all metabolism-related; they were fatty acid degradation, retinol metabolism, and arachidonic acid metabolism. In ICC, on the other hand, additional pathways related to fat digestion and absorption and cholesterol metabolism were identified. Consistent characteristics of such a metabolic landscape were observed across different cohorts. A prognostic risk score model for calculating HCC risk was constructed, consisting of ADH4, ADH6, CYP2C9, CYP4F2, and RDH16. This signature predicts the 3-year survival with an AUC area of 0.708 (95%CI = 0.644 to 0.772). For calculating the risk of ICC, a prognostic risk score model was built upon the expression levels of CYP26A1, NAT2, and UGT2B10. This signature predicts the 3-year survival with an AUC area of 0.806 (95% CI = 0.664 to 0.947). Conclusion HCC and ICC share commonly abrupted pathways associated with the metabolism of fatty acids, retinol, arachidonic acids, and drugs, indicating similarities in their pathogenesis as primary liver cancers. On the flip side, these two types of cancer possess distinctive promising biomarkers for predicting overall survival or potential targeted therapies.

Identification of Ferroptosis-related molecular model and immune subtypes of hepatocellular carcinoma for individual therapy.

Excessive iron accumulation and lipid peroxidation are primary characteristics of ferroptosis in hepatocellular carcinoma (HCC). Ferroptosis inducer combined with immunotherapy has become a new hope for HCC patients. Therefore, the construction and validation of subtype-specific sensitivity to ferroptosis inducer will be helpful for hierarchical management and precise individual therapy. RNA-seq transcriptome and clinical data of HCC patients were extracted from International Cancer Genome Consortium (ICGC) dataset and The Cancer Genome Atlas (TCGA) dataset. Consistency matrix and data clustering of the both cohorts were constructed by 'ConsensusClusterPlus' package. Single-sample gene set enrichment analysis (ssGSEA) analysis was performed to evaluate immune infiltration. Cox analysis was utilized to construct a ferroptosis phenotype-related prognostic model (FRPM) in HCC. The predictive efficiency of the constructed FRPM was evaluated through Kaplan Meier (K-M) survival analyses and Receiver Operating Characteristic (ROC) curves. The expression levels of candidate genes were detected and validated by Real-Time PCR between liver cancer tissues and adjacent non-tumor liver tissues. 45 differentially expressed ferroptosis-related genes (FRGs) were identified between HCC tissues and non-tumor liver tissues. Furthermore, four ferroptosis-associated clusters (FACs) of HCC were established via consensus clustering. Subsequently, we established a FRPM, consisting of four prognostic genes (SLC7A11, SLC1A5, GCLM and SAT1), to evaluate the survival of HCC patients, based on which, patients were divided into high-risk group and low-risk group. The high-risk group exhibited worse survival compared to low-risk group (p< 0.0001 both in TCGA and ICGC cohorts). Patients belong to different FACs or different risk scores showed distinct clinical characteristics. Moreover, in the validation experiment, the transcriptional expression levels of the four prognostic genes were consistent with the results drew from datasets. We revealed a novel FRGs signature, which may provide the molecular characteristic data for effectively prognostic evaluation and potential personalized therapy for HCC patients.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Significance of promoter methylation of multiple tumor suppressor genes in hepatocellular carcinoma

BackgroundMethylation of the promoter at CpG islands is a mechanism of silencing tumor suppressor genes and therefore enhances cancer progression. The study aimed to examine promoter methylation frequencies of five tumor suppressor genes in hepatocellular carcinoma and their implication on the first-year outcome of surgical resection of the tumor. Fifty specimens of hepatocellular carcinoma and the adjacent non-tumorous liver tissue were collected from the surgically resected hepatic tumor. The status of promoter methylation of tumor suppressor genes RASSF1A, CHFR, MGMT, GSTP1, and hMLH1 was investigated using methylation-specific polymerase chain reaction.ResultsThe frequency of promoter methylation of these tumor suppressors genes (TSG) genes in hepatocellular carcinoma was significantly higher than non-tumorous tissue all, P < 0.05, with a methylation rate of 80% in RASSF1A, 70% in CHFR, 46% in GSTP1, 56% in MGMT, and 10% in hMLH1. Methylation of RASSF1A, CHFR, and MGMT promoter genes was significantly associated with decreased first-year postoperative survival and increased recurrence of hepatocellular carcinoma, P < 0.05.ConclusionMethylated RASSF1A, CHRF, and MGMT promoters indicated poor prognosis among patients with hepatocellular carcinoma and may serve as potential prognostic indicators in patients with hepatocellular carcinoma.

PHF21A expression as a biomarker of hepatocellular carcinoma progression and prognosis.

Previous studies have shown that PHF21A is associated with the initiation and progression of various tumors. However, its role in hepatocellular carcinoma (HCC) is still unclear. Thus, this study aimed to determine the expression and clinical significance of PHF21A in HCC. PHF21A expression in 201 liver cancer samples and 129 adjacent normal tissues was detected by immunohistochemistry. The correlation between PHF21A expression and the clinicopathological features and prognosis of HCC was verified in 70 other liver tissue microarray samples. The relationship between PHF21A expression and HCC immune cell infiltration was explored via the Tumor Immune Estimation Resource (TIMER). The mechanism underlying the effect of PHF21A on HCC progression was analyzed by gene set enrichment analysis (GSEA) and protein‒protein interaction (PPI) network analysis. Immunohistochemical staining showed that PHF21A expression in HCC tissue was significantly lower than that in adjacent nontumor liver tissue and was associated with patient sex, tumor size, metastasis, and Edmondson grade (p < 0.05). Kaplan-Meier analysis demonstrated that low PHF21A expression was associated with a poor prognosis, and Cox regression analysis showed that PHF21A was an independent predictor of prognosis. TIMER analysis showed that PHF21A is positively correlated with tumor immune cell infiltration levels. Functional annotation indicated that PHF21A is involved in important pathways, including transcriptional deregulation pathways in cancer. Finally, in vitro experiments confirmed the low expression of PHF21A in HCC cells. PHF21A affects the progression and prognosis of HCC, suggesting that PHF21A may play an important role in monitoring and preventing the development of HCC.

Cellular retinol binding protein-1 inhibits cancer stemness via upregulating WIF1 to suppress Wnt/\u03b2-catenin pathway in hepatocellular carcinoma

BackgroundCRBP-1, a cytosolic chaperone of vitamin A, is identified in a serious number of cancers; however, its biological role in hepatocellular carcinoma (HCC) needs to be further explored. The aim of our present study is to explore the roles and mechanisms of CRBP-1 in regulating liver cancer by using in vitro and in vivo biology approaches.MethodsThe expression level of CRBP-1 was detected using immunohistochemistry in HCC and matching adjacent non-tumorous liver tissues. Following established stable CRBP-1 overexpressed HCC cell lines, the cell growth and tumorigenicity were investigated both in vitro and in vivo. Intracellular retinoic acid was quantified by ELISA. The relationship between CRBP-1 and WIF1 was validated by using dual luciferase and ChIP analyses.ResultsThe low expression of CRBP-1 was observed in HCC tissues compared to the normal liver tissues, while high CRBP-1 expression correlated with clinicopathological characteristics and increased overall survival in HCC patients. Overexpression of CRBP-1 significantly inhibited cell growth and tumorigenicity both in vitro and in vivo. Moreover, overexpression of CRBP-1 suppressed tumorsphere formation and cancer stemness related genes expression in HCC. Mechanically, CRBP-1 inhibited Wnt/β-catenin signaling pathway to suppress cancer cell stemness of HCC. Furthermore, our results revealed that CRBP-1 could increase the intracellular levels of retinoic acid, which induced the activation of RARs/RXRs leading to the transcriptional expression of WIF1, a secreted antagonist of the Wnt/β-catenin signaling pathway, by physically interacting with the region on WIF1 promoter.ConclusionOur findings reveal that CRBP-1 is a crucial player in the initiation and progression of HCC, which provide a novel independent prognostic biomarker and therapeutic target for the diagnosis and treatment of HCC.

Serum Anti-14-3-3 Zeta Autoantibody as a Biomarker for Predicting Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide. Alpha-fetoprotein (AFP) is still the only serum biomarker widely used in clinical settings. However, approximately 40% of HCC patients exhibit normal AFP levels, including very early HCC and AFP-negative HCC; for these patients, serum AFP is not applicable as a biomarker of early detection. Thus, there is an urgent need to identify novel biomarkers for patients for whom disease cannot be diagnosed early. In this study, we screened and identified novel proteins in AFP-negative HCC and evaluated the feasibility of using autoantibodies to those protein to predict hepatocarcinogenesis. First, we screened and identified differentially expressed proteins between AFP-negative HCC tissue and adjacent non-tumor liver tissue using SWATH-MS proteome technology. In total, 2,506 proteins were identified with a global false discovery rate of 1%, of which 592 proteins were expressed differentially with 175 upregulated and 417 downregulated (adjusted p-value <0.05, fold-change FC ≥1.5 or ≤0.67) between the tumor and matched benign samples, including 14-3-3 zeta protein. For further serological verification, autoantibodies against 14-3-3 zeta in serum were evaluated using enzyme-linked immunosorbent, Western blotting, and indirect immunofluorescence assays. Five serial serum samples from one patient with AFP-negative HCC showed anti-14-3-3 zeta autoantibody in sera 9 months before the diagnosis of HCC, which gradually increased with an increase in the size of the nodule. Based on these findings, we detected the prevalence of serum anti-14-3-3 zeta autoantibody in liver cirrhosis (LC) patients, which is commonly considered a premalignant liver disease of HCC. We found that the prevalence of autoantibodies against 14-3-3 zeta protein was 16.1% (15/93) in LC patient sera, which was significantly higher than that in patients with chronic hepatitis (0/75, p = 0.000) and normal human sera (1/60, 1.7%, p = 0.01). Therefore, we suggest that anti-14-3-3 zeta autoantibody might be a biomarker for predicting hepatocarcinogenesis. Further follow-up and research of patients with positive autoantibodies will be continued to confirm the relationship between anti-14-3-3 zeta autoantibody and hepatocarcinogenesis.

Race/ethnicity-associated blood DNA methylation differences between Japanese and European American women: an exploratory study

BackgroundRacial/ethnic disparities in health reflect a combination of genetic and environmental causes, and DNA methylation may be an important mediator. We compared in an exploratory manner the blood DNA methylome of Japanese Americans (JPA) versus European Americans (EUA).MethodsGenome-wide buffy coat DNA methylation was profiled among healthy Multiethnic Cohort participant women who were Japanese (JPA; n = 30) or European (EUA; n = 28) Americans aged 60–65. Differentially methylated CpGs by race/ethnicity (DM-CpGs) were identified by linear regression (Bonferroni-corrected P < 0.1) and analyzed in relation to corresponding gene expression, a priori selected single nucleotide polymorphisms (SNPs), and blood biomarkers of inflammation and metabolism using Pearson or Spearman correlations (FDR < 0.1).ResultsWe identified 174 DM-CpGs with the majority of hypermethylated in JPA compared to EUA (n = 133), often in promoter regions (n = 48). Half (51%) of the genes corresponding to the DM-CpGs were involved in liver function and liver disease, and the methylation in nine genes was significantly correlated with gene expression for DM-CpGs. A total of 156 DM-CpGs were associated with rs7489665 (SH2B1). Methylation of DM-CpGs was correlated with blood levels of the cytokine MIP1B (n = 146). We confirmed some of the DM-CpGs in the TCGA adjacent non-tumor liver tissue of Asians versus EUA.ConclusionWe found a number of differentially methylated CpGs in blood DNA between JPA and EUA women with a potential link to liver disease, specific SNPs, and systemic inflammation. These findings may support further research on the role of DNA methylation in mediating some of the higher risk of liver disease among JPA.