Poor Prognosis In Hepatocellular Carcinoma Research Articles

LncRNA00978 contributes to growth and metastasis of hepatocellular carcinoma cells via mediating microRNA-125b-5p/SOX12 pathway

ABSTRACT As a malignant tumor, HCC (hepatocellular carcinoma) is featured by a high recurrence rate with a poor prognosis. Increasing evidence supports an important role of lincRNAs in HCC. Here, the purpose of the study was to explore the function of LINC00978 (long non-coding RNA00978) in HCC and the underlying mechanisms. LINC00978 expression and its association with the progression of HCC were analyzed using HCC TCGA datasets. LINC00978 expression in tissues was measured using real-time PCR. Then, we knocked down LINC00978 in HCC cells to explore its effect on cellular invasion, proliferation, and migration. Finally, we investigated the potential molecular mechanism of LINC00978 by dual Luciferase reporter assay, FISH (fluorescence in situ hybridization) and RIP (RNA immunoprecipitation). LINC00978 expression was remarkably increased in HCC. A high level of LINC00978 was associated with poor prognosis of HCC. Additionally, LINC00978 silencing could repress the growth and metastasis of HCC cells. Mechanistically, it was revealed that LINC00978 could sponge microRNA-125b-5p and identified SOX12 (SRY-Box Transcription Factor 12) as a direct target gene of microRNA-125b-5p. More importantly, the suppressed effect of LINC00978 silencing on the metastasis and growth of HCC cells could be rescued by miR-125b-5p inhibition and overexpressed SOX12. LINC00978/microRNA-125b-5p/SOX12 axis promoted liver cancer migration, invasion, and proliferation, which could be used as a possible therapeutic target for the treatment of hepatocellular carcinoma.

Identification and Validation of Hub Genes with Poor Prognosis in Hepatocellular Carcinoma by Integrated Bioinformatical Analysis.

BackgroundHepatocellular carcinoma (HCC) is the reason for the world’s second largest cancer-related death. It is clinically valuable to study the molecular mechanisms of HCC occurrence and development for formulating more effective diagnosis and treatment strategies.MethodsThe five microarray data sets GSE45267, GSE101685, GSE84402, GSE62232 and GSE45267 were downloaded from Gene Expression Omnibus (GEO) database, including 165 HCC tissues and 73 normal tissues. Differential expressed genes (DEGs) between HCC tissues and normal tissues were determined by GEO2R. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and the protein–protein interaction network (PPI) network analysis were employed to identify DEGs and to evaluate the clinical significance in prognosis of HCC.ResultsA total of 152 genes differentially expressed in HCC tissues and normal tissues were identified. GO and KEGG functional enrichment analysis revealed that 39 up-regulated genes were mainly enriched in mitosis, cell cycle and oocyte meiosis, while those down-regulated genes (113) were concentrated in exogenous drug catabolism and the metabolism of cytochrome P450 on exogenous drugs. Totally, 19 hub genes were chosen by PPI network and module analysis and verified by The Cancer Genome Atlas (TCGA) database. Finally, 8 hub genes were selected, including CDK1, CYP2C8, CCNB1, AURKA, CYP2C9, BUB1B, MAD2L1 and TTK, which were associated with the overall survival rate of HCC patients.ConclusionThis study presented eight target genes connected to the prognosis of HCC patients. Those mainly exists in cell cycle and drug catabolism, which may be latent targets for clinical treatment.

HCG15 is a hypoxia-responsive lncRNA and facilitates hepatocellular carcinoma cell proliferation and invasion by enhancing ZNF641 transcription

Hypoxia, one of the key features of the hepatocellular carcinoma (HCC) microenvironment, transcriptionally regulates the expression of mRNAs and non-coding RNAs via hypoxia-inducible factors (HIFs), thereby promoting tumor progression. However, hypoxia-responsive long non-coding RNAs (lncRNAs) in HCC required further investigation. We found that HLA complex group 15 (HCG15) was a new hypoxia-related lncRNA in HCC cells. Both hypoxia and HIF prolyl-hydroxylase inhibitor significantly increased HCG15 expression in HCC cells. At the same time, HIF-1α knockdown blocked hypoxia-induced the upregulation of HCG15. TCGA data revealed that HCG15 was markedly overexpressed and closely associated with the poor prognosis of HCC. HCG15 knockdown prominently suppressed the migration, invasion and proliferation of Hep3B and Huh7 cells. HCG15 overexpression markedly enhanced the proliferation and mobility of Huh7 cells. Zinc finger protein 641 (ZNF641) mRNA was frequently overexpressed and positively associated with HCG15 level in HCC samples from the TCGA database. Either HCG15 or upstream transcription factor 1 (USF1) silencing markedly reduced the ZNF641 level in HCC cells. RNA immunoprecipitation assay confirmed the interaction between HCG15 and USF1. Either HCG15 or USF1 knockdown prominently reduced the luciferase activity of reporter plasmid containing ZNF642 promoter. HCG15 knockdown remarkably abolished USF1-activated ZNF641 transcription in Hep3B cells. Finally, we confirmed that restoring ZNF641 expression remarkably abolished the effects of HCG15 knockdown on HCC cell migration, invasion and proliferation. Collectively, our study demonstrated that HCG15 was a new HIF-1 target gene and played a tumor-promoting role in HCC cells by enhancing USF1-mediated ZNF641 transcription.

Knockdown of RSPH14 inhibits proliferation, migration, and invasion and promotes apoptosis of hepatocellular carcinoma via RelA

BackgroundHigh RSPH14 expression appears to be related to poor prognosis of hepatocellular carcinoma (HCC). This study aimed to investigate the possible roles of RSPH14 in the proliferation, apoptosis, and invasion of HCC cells.MethodsThe UALCAN database and Kaplan–Meier Plotter were used to evaluate the expression level and prognostic role of RSPH14 in HCC. Lentiviral vectors containing shRNA against RSPH14 were constructed to transfect the BEL-7404 and SMMC-7721 HCC cell lines. Cell proliferation was investigated by BrdU, MTT, and colony-formation assays. Apoptosis was detected using flow cytometry. Cell migration and invasion were evaluated using the scratch wound-healing and Transwell assays. Immunohistochemistry and western blot were used to determine the expression levels of the proteins. The function of RSPH14 in vivo was evaluated using a xenograft mouse model.ResultsThe expression of RSPH14 was higher in HCC tumor tissues than in adjacent normal tissues and was closely related to unfavorable prognostic factors and poorer survival (all P < 0.05). Knockdown of RSPH14 inhibited the cell proliferation, migration, and invasion of HCC cells and promoted apoptosis (all P < 0.05). Knockdown of RSPH14 inhibited tumor growth in vivo (P < 0.05). RSPH14 knockdown led to decreased expression of RelA (NF-κBp65), CDH2, and AKT1, thereby affecting the functions of the HCC cells (all P < 0.05). RelA overexpression could abate the inhibitory effect of BEL-7404 cell proliferation caused by RSPH14 depletion.ConclusionKnockdown of RSPH14 could decrease cell proliferation, migration, and invasion and increase apoptosis of HCC cells by inhibiting RelA expression. RSPH14 could be a new treatment target for HCC.

ASPM May be Related to the Malignant Progression of Hepatitis B and is Associated With a Poor Prognosis of Hepatocellular Carcinoma.

Background: Hepatitis B virus (HBV) is a causative agent of hepatocellular carcinoma (HCC). Until now, the mechanism behind the progress of hepatitis B fibrosis to HCC remains largely unknown. This study aims to examine the candidate biomarkers and pathways involved in HBV-associated HCC. Methods: Gene expression profiles were retrieved from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the GEO2R tool after which functional enrichment analysis, protein-protein interaction (PPI) analysis, genetic alteration analysis, prognostic analysis, immune infiltration analysis, co-expression genes prediction, and miRNA-gene network construction, and pathway correlation analysis were performed. Results: 22 hub genes were identified, which were all highly expressed in HCC, and overexpression of these genes was all associated with significantly worse survival in HCC patients. More significantly, ASPM also showed increased expression levels in non-tumor tissues with advanced liver fibrosis. With the progression of liver fibrosis and the closer tumor center of HCC, the higher expression of ASPM was identified. ASPM was considered to be the most promising biomarker because it also showed the highest genetic alteration frequency among the hub genes and the expression level of ASPM in HBV (+) HCC tissues was significantly higher than that in HBV (-) HCC tissues. Also, the infiltration levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells were all positively correlated with the expression of ASPM. Conclusion: These findings may help in the development of strategies and candidate drugs for the treatment of HBV-related HCC and improve the effectiveness of personalized treatment in the future. ASPM was upregulated in both hepatitis B cirrhosis and HCC and could be a potential predicting biomarker.

Downregulation of ZC3H13 by miR-362-3p/miR-425-5p is associated with a poor prognosis and adverse outcomes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is notorious for its poor prognosis. Previous studies identified several N6-methyladenosine (m6A)-related genes that play key roles in the initiation and progression of HCC patients. In particular, the N6-methyladenosine RNA methylation regulator ZC3H13 could be a candidate as a novel biomarker and therapeutic target for hepatocellular carcinoma. In HCC, low expression of ZC3H13 was reported, but the molecular reason is unclear. In this study, we performed pan cancer analysis for ZC3H13 expression and prognosis using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data and found that ZC3H13 might be a potential tumor suppressor gene in HCC. Subsequently, miRNAs contributing to ZC3H13 downregulation were identified by a series of in silico analyses, including expression analysis, correlation analysis, and survival analysis. Finally, the miR-362-3p/miR-425-5p-ZC3H13 axis was identified as the most likely upstream miRNA-related pathway of ZC3H13 in HCC. Additionally, miR-362-3p/miR-425-5p mimic and inhibitor results were detected by quantitative real-time PCR (qPCR) analysis and western blotting. We identified an upstream regulatory mechanism of ZC3H13 in HCC, namely, the miR-362-3p/miR-425-5p-ZC3H13 axis. Moreover, the ZC3H13 level was significantly positively associated with tumor immune cell infiltration, biomarkers of immune cells, and immune checkpoint expression. Collectively, our findings elucidated that ncRNA-mediated downregulation of ZC3H13 was correlated with a poor prognosis and tumor immune infiltration in HCC. In conclusion, this study demonstrates that ZC3H13 is a direct target of miR-362-3p/miR-425-5p in liver hepatocellular carcinoma (LIHC) that regulates immune modulation in the microenvironment of LIHC.

Expression of IER3 in hepatocellular carcinoma: clinicopathology, prognosis, and potential regulatory pathways.

BackgroundImmediate early response 3 (IER3) is correlated to the prognosis of several cancers, but the precise mechanisms underlying the regulation by IER3 of the occurrence and development of hepatocellular carcinoma (HCC) remain unknown.MethodsThe expression level of IER3 was examined by using in-house immunohistochemistry (IHC), public gene chip, and public RNA-sequencing (RNA-seq). The standardized mean difference (SMD) was calculated to compare the expression levels of IER3 between HCC patients and controls. The summary receiver operating characteristics (sROC) was plotted to comprehensively understand the discriminatory capability of IER3 between HCC and non-HCC group. The Kaplan–Meier curves and the combined hazard ratios (HRs) were used to determine the prognostic value of IER3 in HCC. Moreover, differentially expressed genes (DEGs) and co-expression genes (CEGs) were used to explored the molecular mechanisms of IER3 underlying HCC. hTFtarget was used to predict the transcription factors (TFs) of IER3. The binding site of TFs and the IER3 promoter region was forecasted using the JASPAR website. The relevant ChIP-seq data were used to determine whether TF peaks were present in the IER3 transcription initiation.ResultsA significantly increased expression of IER3 protein was found in HCC tissue relative to non-HCC tissue as detected by IHC (p < 0.001). Compared to 1,263 cases of non-HCC tissues, IER3 in 1483 cases of HCC tissues was upregulated (SMD = 0.42, 95% confidence interval [CI] [0.09–0.76]). The sROC showed that IER3 had a certain ability at differentiating HCC tissues (area under the curve (AUC) = 0.65, 95% CI [0.61–0.69]). Comprehensive analysis of the effect of IER3 on the prognosis of patients with HCC demonstrated that higher IER3 expression was associated with poor prognosis in HCC (HRs = 1.30, 95% CI [1.03–1.64]). Pathway enrichment analysis revealed that IER3-related genes were mostly enriched in the PI3K-Akt signaling pathway, cancer-related signaling pathways, the p53 signaling pathway, and other signaling pathways. Regulatory factor X5 (RFX5) was identified as a possible regulator of IER3-related TF.ConclusionIER3 may be a potential prognostic marker for HCC. The molecular mechanisms of IER3 in HCC warrant further study.

Increased CDCA2 Level Was Related to Poor Prognosis in Hepatocellular Carcinoma and Associated With Up-Regulation of Immune Checkpoints.

BackgroundCell division cycle-associated protein 2 (CDCA2) is a member of cell cycle-related proteins. CDCA2 plays a role in the regulation of protein phosphatase 1(PP1) γ-dependent DNA damage response (DDR) and H3 phosphorylation. CDCA2 promotes the tumorigenesis and development of several types of cancers by promoting the proliferation of tumor cells. However, the relationship between CDCA2 expression and the clinicopathological characteristics of hepatocellular carcinoma (HCC) is unknown.MethodsGene expression information and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. The expression of CDCA2 and its correlation to clinical characteristics in HCC were analyzed. The expression level of CDCA2 was validated in HCC cell lines. The relationship between CDCA2 expression and the survival of patients with HCC was analyzed by using Kaplan–Meier method. The prognostic value of CDCA2 in HCC was estimated by Cox regression analysis. The expression difference of CDCA2 between HCC and normal tissues and its correlation to survival were verified in independent datasets. Gene set enrichment analysis (GSEA) was used to screen the CDCA2-related signaling pathways.ResultsCell division cycle-associated protein 2 expression was upregulated in HCC tissues (p < 0.001) and increased CDCA2 was correlated to increased T stage, pathologic stage, histologic grade, and alpha-fetoprotein (AFP) level (p < 0.001). In addition, CDCA2 was overexpressed in HCC cell lines HepG2 and LM3. High CDCA2 expression level was associated with poor overall survival [hazard ratio (HR) = 1.69; 95% CI, 1.20–1.40, p = 0.003], disease specific survival (HR = 1.73; 95% CI, 1.11–2.71, p = 0.016), and progress free interval (HR = 1.74; 95% CI, 1.30–2.34, p < 0.001). Overexpression of CDCA2 and its correlation to poor survival in HCC were verified in Gene Expression Omnibus (GEO) datasets and Kaplan–Meier plotter database. Increased CDCA2 expression was associated with upregulation of PD-L1 (Spearman's coefficient = 0.207, p < 0.001), PD-L2 (Spearman coefficient's = 0.118, p < 0.05), and CTLA4 (Spearman's coefficient = 0.355, p < 0.001). GSEA showed that homologous recombination pathway, insulin signaling pathway, mitogen-activated protein kinase (MAPK) pathway, mismatch repair pathway, mechanistic target of rapamycin (mTOR) pathway, Notch pathway, T cell receptor pathway, toll like receptor pathway, and WNT pathway were enriched in CDCA2 high expression phenotype.ConclusionCell division cycle-associated protein 2 may serve as an independent biomarker for poor prognosis in HCC and increased CDCA2 expression was associated with upregulation of immune checkpoints.

Prognostic and biological role of the N-Myc downstream-regulated gene family in hepatocellular carcinoma

BACKGROUNDThe N-Myc downstream-regulated gene (NDRG) family is comprised of four members (NDRG1-4) involved in various important biological processes. However, there is no systematic evaluation of the prognostic of the NDRG family in hepatocellular carcinoma (HCC). AIMTo analyze comprehensively the biological role of the NDRG family in HCC.METHODSThe NDRG family expression was explored using The Cancer Genome Atlas. DNA methylation interactive visualization database was used for methylation analysis of the NDRG family. The NDRG family genomic alteration was assessed using the cBioPortal. Single-sample Gene Set Enrichment Analysis was used to determine the degree of immune cell infiltration in tumors. RESULTSNDRG1 and NDRG3 were up-regulated in HCC, while NDRG2 was down-regulated. Consistent with expression patterns, high expression of NDRG1 and NDRG3 was associated with poor survival outcomes (P < 0.05). High expression of NDRG2 was associated with favorable survival (P < 0.005). An NDRG-based signature that statistically stratified the prognosis of the patients was constructed. The percentage of genetic alterations in the NDRG family varied from 0.3% to 11.0%, and the NDRG1 mutation rate was the highest. NDRG 1-3 expression was associated with various types of infiltrated immune cells. Gene ontology analysis revealed that organic acid catabolism was the most important biological process related to the NDRG family. Gene Set Enrichment Analysis showed that metabolic, proliferation, and immune-related gene sets were enriched during NDRG1 and NDRG3 high expression and NDRG2 low expression.CONCLUSIONOverexpression of NDRG1 and NDRG3 and down-expression of NDRG2 are correlated with poor overall HCC prognosis. Our results may provide new insights into the indispensable role of NDRG1, 2, and 3 in the development of HCC and guide a promising new strategy for treating HCC.

High Expression of RRM1 Mediated by ncRNAs Correlates with Poor Prognosis and Tumor Immune Infiltration of Hepatocellular Carcinoma.

IntroductionHepatocellular carcinoma (HCC) is one of several tumors with poor prognosis and causes a significant social burden. A growing number of studies have shown that RRM1 plays a crucial role in the development and progression of multiple human cancers. However, the specific role and mechanism of RRM1 have not been fully defined in HCC.MethodsTCGA and GTEx data were used for the first time to conduct a pan-cancer analysis of RRM1 expression and prognosis, and identified RRM1 as a possible potential oncogene in HCC. At the same time, a combination of analyses (including expression analysis, correlation analysis or survival analysis) identified non-coding RNAs (ncRNAs) that contribute to RRM1 overexpression.ResultsMIR4435-2HG/miR-22-3p and SNHG6/miR-101-3p were identified as the most promising RRM1 upstream ncRNA-related pathways in HCC. In addition, RRM1 levels were significantly and positively correlated with tumor immune cell infiltration, immune cell biomarker or immune checkpoint expression.ConclusionThese results suggest that high expression of RRM1 mediated by ncRNAs is associated with poor prognosis and tumor immune infiltration in HCC.

ITRAQ-Based Proteome Profiling of Differentially Expressed Proteins in Insulin-Resistant Human Hepatocellular Carcinoma.

Recently, the incidences of insulin resistance (IR) and IR-related complications have increased throughout the world, which also associate with poor prognosis in hepatocellular carcinoma (HCC). Numerous studies had been focused on the role of IR in tumorigenesis and prognosis of HCC. The proteomic analysis of IR related hepatocellular carcinoma had not been reported by now. In the present study, 196 differentially expressed proteins (DEPs) were identified between insulin resistant HepG2 cells and their parental cells, of which 109 proteins were downregulated and 87 proteins were upregulated. Bioinformatics analysis indicated that these DEPs were highly enriched in process of tumorigenesis and tumor progression. PPI network analysis showed that SOX9, YAP1 and GSK3β as the key nodes, were involved in Wnt and Hippo signaling pathways. Survival analysis revealed that high expression of SOX9 and PRKD3 were strongly associated with reduced patient survival rate. parallel reaction monitoring (PRM) and Western blot analysis were applied to verify the protein level of these four key nodes mentioned above, which showed the same trend as quantified by isobaric tags for relative and absolute quantitation (iTRAQ) and confirmed the reliability of our Proteome Profiling analysis. Our results indicated that IR related dysregulation of protein expression might participated in tumorigenesis and malignant phenotype of hepatocarcinoma cells.

A Comprehensive Prognostic Analysis of POLD1 in Hepatocellular Carcinoma

BackgroundDNA polymerase delta 1 catalytic subunit (POLD1) plays a key role in DNA replication and damage repair. A defective DNA proofreading function caused by POLD1 mutation contributes to carcinogenesis, while POLD1 overexpression predicts poor prognosis in cancers. However, the effect of POLD1 in hepatocellular carcinoma (HCC) is not well-understood.MethodsExpression patterns of POLD1 were evaluated in TCGA and the HPA databases. Kaplan-Meier curves and Cox regression were used to examine the prognostic value of POLD1. The prognostic and predictive value of POLD1 was further validated by another independent cohort from ICGC database. The influences of DNA copy number variation, methylation and miRNA on POLD1 mRNA expression were examined. The correlation between infiltrating immune cells and POLD1 expression was analyzed. GO and KEGG enrichment analyses were performed to detect biological pathways associated with POLD1 expression in HCC.ResultsPOLD1 was overexpressed in HCC (n = 369) compared with adjacent normal liver (n = 50). POLD1 upregulation was significantly correlated with positive serum AFP and advanced TNM stage. Kaplan–Meier and multivariate analyses suggested that POLD1 overexpression predicts poor prognosis in HCC. DNA copy gain, low POLD1 methylation, and miR‑139-3p downregulation were associated with POLD1 overexpression. Besides, POLD1 expression was associated with the infiltration levels of dendritic cell, macrophage, B cell, and CD4 + T cell in HCC. Functional enrichment analysis suggested “DNA replication”, “mismatch repair” and “cell cycle” pathways might be involved in the effect of POLD1 on HCC pathogenesis. Additionally, POLD1 mRNA expression was significantly associated with tumor mutation burden, microsatellite instability, and prognosis in various tumors.ConclusionsPOLD1 may be a potential prognostic marker and promising therapeutic target in HCC.

An Integrated Analysis of the Identified PRPF19 as an Onco-immunological Biomarker Encompassing the Tumor Microenvironment, Disease Progression, and Prognoses in Hepatocellular Carcinoma.

Background: Targeting the mRNA splicing process has been identified as a therapeutic strategy for human cancer. PRPF19 is an RNA binding protein that is involved in pre-mRNA processing and repairing DNA damage; the aberrant expression of PRPF19 is potentially associated with carcinogenesis. However, the biological role of PRPF19 in hepatocellular carcinoma (HCC) is still elusive. Methods: Data obtained from TCGA, Oncomine, and GEO were used to investigate the PRPF19 expression level and its role in tumor immune infiltration, prognosis, and the tumor progression of cohorts from HCC. Using various databases and tools (UALCAN, TIMER, TISMO, and PathCards), we presented the potential mechanisms of PFPF19 upregulation, PRPF19-related pathways, and its biological functions in liver cancer. Results: For HCC, PRPF19 expression was found upregulated both in single tumor cells and tissues. Furthermore, the increased expression of PRPF19 was significantly correlated to clinical characteristics: advanced stage, vascular invasion, high AFP, and poor prognosis of HCC. According to the tumor-immunological analysis, we found that PRPF19 is positively correlated with infiltrating myeloid-derived suppressor cells (MDSCs). Moreover, the microenvironment of HCC tissues with high expression of PRPF19 is highly immunosuppressive (lower T-lymphocytes, multiple immune checkpoints upregulated). Patients with high expression of PRPF19 and high MDSCs had a worse survival prognosis as well. TP53 mutation may have a positive effect on PRPF19 expression via decreased promoter methylation of PRPF19. By TF-mRNA network analysis, key transcription factors (TFs) in TC-NER and PCS pathways (PRPF19 involved) were identified. Conclusion: This work implied that PRPF19 is associated with tumor immune evasion and progression, and serves as a prognostic marker for worse clinical outcomes with HCC. Thus, this critical regulator could serve as a potential therapeutic target of HCC.

PARG inhibition limits HCC progression and potentiates the efficacy of immune checkpoint therapy

Although the treatment of hepatocellular carcinoma (HCC) has been revolutionized by the advent of effective systemic therapies, the prognosis of patients with HCC remains dismal. Herein, we examined the pathophysiological role of PARG and assessed the utility of targeting dePARylation for HCC therapy. The oncogenic function of PARG was evaluated in 2 orthotopic xenograft models and a Pargflox/flox mice model. The therapeutic efficacy of PARG inhibitors in combination with an anti-PD-1 antibody were assessed in murine orthotopic models. Microarray analysis was used to evaluate the pathological relevance of the PARG/DDB1/c-Myc/MMR axis. High PARG expression was strongly associated with poor HCC prognosis. Hepatocyte-specific PARG deletion significantly impaired liver tumorigenesis. PARG promoted HCC growth and metastasis through DDB1-dependent modulation of c-Myc. Specifically, PARG dePARylated DDB1 and consequently promoted DDB1 autoubiquitination, thus stabilizing the c-Myc protein in HCC cells. PARG downregulation attenuated c-Myc-induced MMR expression and PARG deficiency was correlated with a favorable prognosis in patients with HCC treated with anti-PD-1-based immunotherapy. In addition, PARG inhibitors could act in synergy with anti-PD-1 antibodies in orthotopic mouse models. PARG can act as an oncogene in HCC by modulating PARG/DDB1/c-Myc signaling and could be used as a biomarker to identify patients with HCC who may benefit from anti-PD-1 treatment. Our findings suggest that co-inhibition of PARG and PD-1 is an effective novel combination strategy for patients with HCC. The increase in deaths due to hepatocellular carcinoma (HCC) is a growing concern, with the mechanisms responsible for HCC development still incompletely understood. Herein, we identify a novel mechanism by which the protein PARG contributes to HCC development. Inhibition of PARG increased the efficacy of anti-PD-1 therapy (a type of immunotherapy) in HCC. These findings support the future clinical development of PARG inhibitors, potentially in combination with anti-PD-1 inhibitors.

Ligand-activated PPAR\u03b4 expression promotes hepatocellular carcinoma progression by regulating the PI3K-AKT signaling pathway

BackgroundPeroxisome proliferator-activated receptor-beta/delta (PPARδ) was considered as the key regulator involved in the evolution of various tumors. Given that PPARδ potential role in hepatocellular carcinoma (HCC) is still obscure, we comprehensively assessed its expression pattern, prognosis, functions and correlation with tumor microenvironment in HCC using public database data and in vitro studies.MethodsTranscriptional data and clinical data in the TCGA and GEO database were analyzed in R software. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry were used to detect the expression level of related RNA and proteins. The malignant biological characteristics were explored by cell counting Kit-8 (CCK8), 5-Ethynyl-2ʹ-deoxyuridine (EdU) assay and wound healing assay.ResultsOur results illustrated that PPARδ expression was significantly higher in HCC tissues and HCC cell lines. Elevated expression of PPARδ suggested poor clinical staging and prognosis in HCC. Ligand-activated PPARδ expression promoted the proliferation and invasion of HCC cells via PDK1/AKT/GSK3β signaling pathway. The expression of PPARδ was closely related to the HCC tumor microenvironment.ConclusionsPPARδ plays an important part in HCC progression, penetrating investigation of the related regulatory mechanism may shed light upon further biological and pharmacological value.

DDR1 promotes hepatocellular carcinoma metastasis through recruiting PSD4 to ARF6

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family, and its ligand is collagen. Previous studies demonstrated that DDR1 is highly expressed in many tumors. However, its role in hepatocellular carcinoma (HCC) remains obscure. In this study, we found that DDR1 was upregulated in HCC tissues, and the expression of DDR1 in TNM stage II-IV was higher than that in TNM stage I in HCC tissues, and high DDR1 expression was associated with poor prognosis. Gene expression analysis showed that DDR1 target genes were functionally involved in HCC metastasis. DDR1 positively regulated the migration and invasion of HCC cells and promoted lung metastasis. Human Phospho-Kinase Array showed that DDR1 activated ERK/MAPK signaling pathway. Mechanically, DDR1 interacted with ARF6 and activated ARF6 through recruiting PSD4. The kinase activity of DDR1 was required for ARF6 activation and its role in metastasis. High expression of PSD4 was associated with poor prognosis in HCC. In summary, our findings indicate that DDR1 promotes HCC metastasis through collagen induced DDR1 signaling mediated PSD4/ARF6 signaling, suggesting that DDR1 and ARF6 may serve as novel prognostic biomarkers and therapeutic targets for metastatic HCC.

Targeting Mitochondrial COX-2 Enhances Chemosensitivity via Drp1-Dependent Remodeling of Mitochondrial Dynamics in Hepatocellular Carcinoma.

Simple SummaryNew therapeutic strategies are urgently needed to improve the anti-cancer effect for hepatocellular carcinoma (HCC). Overexpression of cyclooxygenase-2 (COX-2) is found in several types of cancers and correlates with a poor prognosis. However, it remains unclear how the mitochondrial translocation of COX-2 is involved in mitochondrial dynamics and sensitizes HCC cells to multipattern anti-tumor therapy. We explored the impact of targeting mitochondrial COX-2 (mito-COX-2) intervention toward mitochondrial dynamics on platinum-based chemotherapeutics in HCC cells and xenograft nude mouse models. Our study indicates that the mito-COX-2 represents a candidate predictive biomarker and potential target to regulate anti-cancer sensitization of HCC, and possibly for other types of COX-2-high-expression cancers.Mitochondria are highly dynamic organelles and undergo constant fission and fusion, which are both essential for the maintenance of cell physiological functions. Dysregulation of dynamin-related protein 1 (Drp1)-dependent mitochondrial dynamics is associated with tumorigenesis and the chemotherapeutic response in hepatocellular carcinoma (HCC). The enzyme cyclooxygenase-2 (COX-2) is overexpressed in most cancer types and correlates with a poor prognosis. However, the roles played by the translocation of mitochondrial COX-2 (mito-COX-2) and the interaction between mito-COX-2 and Drp1 in chemotherapeutic responses remain to be elucidated in the context of HCC. Bioinformatics analysis, paired HCC patient specimens, xenograft nude mice, immunofluorescence, transmission electron microscopy, molecular docking, CRISPR/Cas9 gene editing, proximity ligation assay, cytoplasmic and mitochondrial fractions, mitochondrial immunoprecipitation assay, and flow cytometry analysis were performed to evaluate the underlying mechanism of how mito-COX-2 and p-Drp1Ser616 interaction regulates the chemotherapeutic response via mitochondrial dynamics in vitro and in vivo. We found that COX-2 and Drp1 were frequently upregulated and confer a poor prognosis in HCC. We also found that the proportion of mito-COX-2 and p-Drp1Ser616 was increased in HCC cell lines. In vitro, we demonstrated that the enhanced mitochondrial translocation of COX-2 promotes its interaction with p-Drp1Ser616 via PTEN-induced putative kinase 1 (PINK1)-mediated Drp1 phosphorylation activation. This increase was associated with higher colony formation, cell proliferation, and mitochondrial fission. These findings were confirmed by knocking down COX-2 in HCC cells using CRISPR/Cas9 technology. Furthermore, inhibition of Drp1 using pharmacologic inhibitors (Mdivi-1) or RNA interference (siDNM1L) decreased mito-COX-2/p-Drp1Ser616 interaction-mediated mitochondrial fission, and increased apoptosis in HCC cells treated with platinum drugs. Moreover, inhibiting mito-COX-2 acetylation with the natural phytochemical resveratrol resulted in reducing cell proliferation and mitochondrial fission, occurring through upregulation of mitochondrial deacetylase sirtuin 3 (SIRT3), which, in turn, increased the chemosensitivity of HCC to platinum drugs in vitro and in vivo. Our results suggest that targeting interventions to PINK1-mediated mito-COX-2/p-Drp1Ser616-dependent mitochondrial dynamics increases the chemosensitivity of HCC and might help us to understand how to use the SIRT3-modulated mito-COX-2/p-Drp1Ser616 signaling axis to develop an effective clinical intervention in hepatocarcinogenesis.

ROCK2 Polymorphism and Expression Contribute to Increased Susceptibility and Poor Prognosis in Hepatocellular Carcinoma.

AimTo analyze the association between ROCK2 gene polymorphism/expression and susceptibility/prognosis of hepatocellular carcinoma (HCC).MethodsPeripheral venous blood from 204 hepatocellular carcinoma patients (HCC group), 242 chronic hepatitis B patients (CHB group), 215 liver cirrhosis patients (LC group) and 201 healthy volunteers (normal group) were collected. ROCK2 gene (including the rs9808232 locus) was amplified by PCR and the products were sequenced. Tumor tissues from patients with HCC and liver tissues from CHB, LC and normal groups were collected, and mRNA and protein expression of ROCK2 gene in liver tissues were tested by RT-qPCR and immunofluorescence, respectively.ResultsCompared with the normal group, the mRNA and protein expression levels of ROCK2 in the HCC group increased significantly (P < 0.05), but there are no obvious increases in the CHB and LC groups (P > 0.05). ROCK2 gene rs9808232 polymorphism was associated with an increased susceptibility of HCC, and genotypes AC and CC may be risk factors for HCC (P < 0.001). There was a significant association between the ROCK2 gene rs9808232 polymorphism and the route of metastasis in HCC patients (P < 0.05). According to the Kaplan–Meier analysis, the overall survival of patients with AC and CC genotypes of ROCK2 rs9808232 was much shorter than that of patients with AA genotype (median overall survival, 25.5 months vs 16.0 months; Log rank P = 0.006). The overall survival time of patients with high ROCK2 expression is much shorter than that of patients with low ROCK2 expression (median overall survival time, 15.0 months vs 20.5 months; Log rank P = 0.008). Compared with genotype AA, the ROCK2 protein levels of genotypes AC and CC were distinctly increased in the HCC group, especially genotype CC (P< 0.05).ConclusionROCK2 gene rs9808232 polymorphism may contribute to its expression in liver tissue, which may increase the susceptibility and poor prognosis of HCC.

ACADL Functions as a Tumor Suppressor in Hepatocellular Carcinoma Metastasis by Inhibiting Matrix Metalloproteinase 14.

High aggressiveness is the main reason for the poor prognosis of hepatocellular carcinoma (HCC) patients. However, its molecular mechanisms still remain largely unexplored. ACADL, a mitochondrial enzyme that facilitates the primary regulated step in mitochondrial fatty acid oxidation, plays a role in HCC growth inhibition. However, the function of ACADL in tumor metastasis is not well elucidated. We found that the reduced expression of ACADL is closely associated with the loss of tumor encapsulation, extrahepatic metastasis, and poor prognosis in HCC patients. Upregulation of ACADL significantly inhibited HCC migration and invasion ability. Whereas knockdown of ACADL markedly enhanced cell invasive capability. Expression of matrix metalloproteinase-14 (MMP14) was negatively associated with the content of ACADL in HCC specimens. MMP14-positive patients with a low expression of ACADL showed worse outcome. Treatment with MMP14 agonist reversed the inhibitory effect of ACADL on HCC metastasis. In addition, ACADL negatively regulated MMP14 expression by inhibiting the STAT3 signaling pathway, as the sustained activation of STAT3 effectively restored the level of MMP14 in ACADL-overexpressed cells. Collectively, these findings disclose that ACADL represses HCC metastasis via STAT3-MMP14 pathway. This study may propose a promising strategy for the precise treatment of metastatic HCC patients.

Bioinformatics screening the novel and promising targets of curcumin in hepatocellular carcinoma chemotherapy and prognosis

BackgroundThe aim of present study was to screen the novel and promising targets of curcumin in hepatocellular carcinoma diagnosis and chemotherapy.MethodsPotential targets of curcumin were screened from SwissTargetPrediction, ParmMapper and drugbank databases. Potential aberrant genes of hepatocellular carcinoma were screened from Genecards databases. Fifty paired hepatocellular carcinoma patients’ gene expression profiles from the GEO database were used to test potential targets of curcumin. Besides, GO analysis, KEGG pathway enrichment analysis and PPI network construction were used to explore the underlying mechanism of candidate hub genes. ROC analysis and Kaplan-Meier analysis were used to evaluate the diagnostic and prognostic value of candidate hub genes, respectively. Real-time PCR was used to verify the results of bioinformatics analysis.ResultsBioinformatics analysis results suggested that AURKA, CDK1, CCNB1, TOP2A, CYP2B6, CYP2C9, and CYP3A4 genes served as candidate hub genes. AURKA, CDK1, CCNB1 and TOP2A were significantly upregulated and correlated with poor prognosis in hepatocellular carcinoma, AUC values of which were 95.7, 96.9, 98.1 and 96.1% respectively. There was not significant correlation between the expression of CYP2B6 and prognosis of hepatocellular carcinoma, while CYP2C9 and CYP3A4 genes were significantly downregulated and correlated with poor prognosis in hepatocellular carcinoma. AUC values of CYP2B6, CYP2C9, and CYP3A4 were 96.0, 97.0 and 88.0% respectively. In vitro, we further confirmed that curcumin significantly downregulated the expression of AURKA, CDK1, and TOP2A genes, while significantly upregulated the expression of CYP2B6, CYP2C9, and CYP3A4 genes.ConclusionsOur results provided a novel panel of AURKA, CDK1, TOP2A, CYP2C9, and CYP3A4 candidate genes for curcumin related chemotherapy of hepatocellular carcinoma.