Paired Hepatocellular Carcinoma Research Articles

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy of Paired Clinical Liver Tissue Samples from Hepatocellular Cancer and Surrounding Region.

The global burden of liver cancer is increasing. Timely diagnosis is important for optimising the limited available treatment options. Understanding the metabolic consequences of hepatocellular carcinoma (HCC) may lead to more effective treatment options. We aimed to document metabolite differences between HCC and matched surrounding tissues of varying aetiology, obtained at the time of liver resection, and to interpret metabolite changes with clinical findings. High-resolution magic angle spinning nuclear magnetic resonance (HRMAS-NMR) spectroscopy analyses of N = 10 paired HCC and surrounding non-tumour liver tissue samples were undertaken. There were marked HRMAS-NMR differences in lipid levels in HCC tissue compared to matched surrounding tissue and more subtle changes in low-molecular-weight metabolites, particularly when adjusting for patient-specific variability. Differences in lipid-CH3, lipid-CH2, formate, and acetate levels were of particular interest. The obvious differences in lipid content highlight the intricate interplay between metabolic adaptations and cancer cell survival in the complex microenvironment of liver cancer. Differences in formate and acetate might relate to bacterial metabolites. Therefore, documentation of metabolites in HCC tissue according to histology findings in patients is of interest for personalised medicine approaches and for tailoring targeted treatment strategies.

DNA damages in hepatocytes are amended by an inflammation-driven rescue repair mechanism in chronic hepatitis B

BackgroundOur previous study has shown that intrahepatic necroinflammation favors the eliminations of HBV integration and clonal hepatocytes. Here, the effect of inflammation on host DNA damage eliminations in liver biopsy tissues from patients with chronic hepatitis B (CHB) was further investigated. MethodsDNA damage markers, histone γ-H2AX and phosphorylated heterochromatin protein 1γ (p-HP1γ), and senescent marker p21 were detected using immunohistochemical and immunofluorescent assays in liver biopsy samples from 69 CHB patients and 12 liver cirrhosis (LC) patients. Twenty paired hepatocellular carcinoma (HCC) surgical samples were used as controls. ResultsBoth γ-H2AX and p-HP1γ were sensitively detected in nuclear and cytoplasmic/nuclear patterns. Nuclear γ-H2AX was superior as a DNA damage marker in hepatocytes. The level of nuclear γ-H2AX in CHB, comparable to those in LC and HCC, was correlated with liver fibrosis and coexisted with the senescent marker p21. However, hepatocytes carried an alleviated level of DNA damages, which was associated with the level of cytoplasmic γ-H2AX. Cytoplasmic γ-H2AX chiefly occurred in hepatocytes near necroinflammatory foci, was correlated with liver inflammation and usually indicated the decrease or disappearance of nuclear γ-H2AX. The lack of cytoplasmic γ-H2AX together with the high level of nuclear γ-H2AX was associated with the progression from large cell changes/dysplasia to small cell changes/dysplasia. ConclusionsHepatocytes in CHB already carry massive DNA damages and undergo cellular senescence. The DNA damages in those senescent hepatocytes are histopathologically demonstrated to be amended by a novel cytoplasmic γ-H2AX-indicated and inflammation-driven rescue repair mechanism, which may be involved in hepatocarcinogenesis if it works improperly.

A novel risk scoring system predicts overall survival of hepatocellular carcinoma using cox proportional hazards machine learning method

BackgroundRobust and practical prognosis prediction models for hepatocellular carcinoma (HCC) patients play crucial roles in personalized precision medicine. Material and methodsWe recruited two independent HCC cohorts (discovery cohort and validation cohort), totally consisting of 222 HCC patients undergone surgical resection. We quantified the expressions of immune-related proteins (CD8, CD68, CD163, PD-1 and PD-L1) in paired HCC tissues and non-tumor liver tissues from these HCC patients using immunohistochemistry (mIHC) assays. We constructed the HCC prognosis prediction model using five different machine learning methods based on the patients in the discovery cohort, such as Cox proportional hazards (CoxPH). ResultsWe identified 19 features that were associated with overall survival of HCC patients in the discovery cohort (p < 0.1), such as immune-related features CD68+ and CD8+ cell infiltration. We constructed five HCC prognosis prediction models using five different machine learning methods. Among the five different machine learning models, the CoxPH model achieved the best performance (area under the curve [AUC], 0.839; C-index, 0.779). According to the risk score from CoxPH model, we divided HCC patients into high-risk group/low-risk group. In both discovery cohort and validation cohort, the patients in low-risk group showed longer overall survival compared with those in high-risk group (p = 1.8 × 10−7 and 3.4 × 10−5, respectively). Moreover, our novel scoring system efficiently predicted the 6, 12, and 18 months survival rate of HCC patients with AUC >0.75 in both discovery cohort and validation cohort. In addition, we found that the scoring system could also distinguish the patients with high/low risks of relapse in both discovery cohort and validation cohort (p = 0.00015 and 0.00012). ConclusionThe novel CoxPH-based risk scoring model on clinical, laboratory-testing and immune-related features showed high prediction efficiencies for overall survival and recurrence of HCCs undergone surgical resection. Our results may be helpful to optimize clinical follow-up or therapeutic interventions.

Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma

ABSTRACT Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.

Transferrin receptor 1 promotes hepatocellular carcinoma progression and metastasis by activating the mTOR signaling pathway.

Aberrant iron metabolism is commonly observed in multiple tumor types, including hepatocellular carcinoma (HCC). However, as the key regulator of iron metabolism involved in iron absorption, the role of transferrin receptor (TFRC) in HCC remains elusive. The mRNA and protein expression of TFRC were evaluated in paired HCC and adjacent non-tumor specimens. The correlation between TFRC level and clinicopathological features or prognostic significance was also analyzed. The role of TFRC on biological functions was finally studied in vitro and in vivo. The TFRC level was remarkably upregulated in HCC tissues compared to paired peritumor tissues. Overexpressed TFRC positively correlated with serum alpha-fetoprotein, carcinoembryonic antigen, and poor tumor differentiation. Multivariate analysis demonstrated that upregulated TFRC was an independent predictive marker for poorer overall survival and disease-free survival in HCC patients. Loss of TFRC markedly impaired cell proliferation and migration in vitro and notably suppressed HCC growth and metastasis in vivo, while overexpression of TFRC performed an opposite effect. Mechanistically, the mTOR signaling pathway was downregulated with TFRC knockdown, and the mTOR agonist MHY1485 completely reversed the biological inhibition in HCC cells caused by TFRC knockdown. Furthermore, exogenous ferric citrate (FAC) or iron chelator reversed the changed biological functions and signaling pathway expression of HCC cells caused by TFRC knockdown or overexpression, respectively. Our study indicates that TFRC exerts an oncogenic role in HCC and may become a promising therapeutic target to restrain HCC progression.

Genome-Wide Extrachromosomal Circular DNA Profiling of Paired Hepatocellular Carcinoma and Adjacent Liver Tissues.

Hepatocellular carcinoma (HCC) develops through multiple mechanisms. While recent studies have shown the presence of extrachromosomal circular DNA (eccDNA) in most cancer types, the eccDNA expression pattern and its association with HCC remain obscure. We aimed to investigate this problem. The genome-wide eccDNA profiles of eight paired HCC and adjacent non-tumor tissue samples were comprehensively elucidated based on Circle-seq, and they were further cross-analyzed with the RNA sequencing data to determine the association between eccDNA expression and transcriptome dysregulation. A total of 60,423 unique eccDNA types were identified. Most of the detected eccDNAs were smaller than 1 kb, with a length up to 182,363 bp and a mean sizes of 674 bp (non-tumor) and 813 bp (tumor), showing a greater association with gene-rich rather than with gene-poor regions. Although there was no statistical difference in length and chromosome distribution, the eccDNA patterns between HCC and adjacent non-tumor tissues showed significant differences at both the chromosomal and single gene levels. Five of the eight HCC tissues showed significantly higher amounts of chromosome 22-derived eccDNA expression compared to the non-tumor tissue. Furthermore, two genes, SLC16A3 and BAIAP2L2, with a higher transcription level in tumor tissues, were related to eccDNAs exclusively detected in three HCC samples and were negatively associated with survival rates in HCC cohorts from public databases. These results indicate the existence and massive heterogeneity of eccDNAs in HCC and adjacent liver tissues, and suggest their potential association with dysregulated gene expression.

RNF149 Promotes HCC Progression through Its E3 Ubiquitin Ligase Activity.

Hepatocellular carcinoma (HCC) accounts for over 80% of cases among liver cancer, with high incidence and poor prognosis. Thus, it is of valuable clinical significance for discovery of potential biomarkers and drug targets for HCC. In this study, based on the proteomic profiling data of paired early-stage HCC samples, we found that RNF149 was strikingly upregulated in tumor tissues and correlated with poor prognosis in HCC patients, which was further validated by IHC staining experiments of an independent HCC cohort. Consistently, overexpression of RNF149 significantly promoted cell proliferation, migration, and invasion of HCC cells. We further proved that RNF149 stimulated HCC progression via its E3 ubiquitin ligase activity, and identified DNAJC25 as its new substrate. In addition, bioinformatics analysis showed that high expression of RNF149 was correlated with immunosuppressive tumor microenvironment (TME), indicating its potential role in immune regulation of HCC. These results suggest that RNF149 could exert protumor functions in HCC in dependence of its E3 ubiquitin ligase activity, and might be a potential prognostic marker and therapeutic target for HCC treatment.

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.

Robust analysis of a novel PANoptosis-related prognostic gene signature model for hepatocellular carcinoma immune infiltration and therapeutic response

PANoptosis, an interplay between pyroptosis, apoptosis, and necroptosis, is deeply involved in cancer development and immunity. However, the influence of PANoptosis in hepatocellular carcinoma (HCC) remains to be further investigated. The differentially expressed PANoptosis-related genes (PANRGs) was screened in The Cancer Genome Atlas (TCGA) database. Accordingly, mutation, bioinformatics, and consensus clustering analyses were performed. Then, a prognostic risk model was developed by least absolute shrinkage and selection operator (LASSO) Cox regression. Furthermore, the prognostic value, immunity correlation and therapeutic response prediction ability of risk model were explored. A total of 18 PANRGs were differently expressed in the TCGA-HCC cohort and were mainly involved in cancer- and cell death-related signal pathways. Using unsupervised clustering method, we identified two PANRGs-mediated clustering patterns. The remarkable differences between the two clusters on overall survival (OS) and clinical features were demonstrated respectively. Based on the five-gene prognostic risk model, the calculated PANRG-scores were used to categorize the subgroups as high- and low-risk. Notably, the high-risk subgroup had a dismal prognosis and exhibited much lower immune infiltration levels of mast cells, nature killer cells and pDCs, but higher levels of aDCs, iDCs and Treg cells than those in the low-risk subgroup. Furthermore, we constructed a reliable nomogram combining clinical traits and PANRG-score to predict the OS of HCC patients. The significantly negative correlation between PANoptosis and tumor mutation burden (TMB), ferroptosis were revealed. In drug sensitivity analysis, the high-risk subgroup had a considerably lower TIDE score, suggesting a preferable response to immunotherapy, and may be more sensitive to Tipifarnib, Imatinib, Doxorubicin, and Gemcitabine. The upregulated mRNA expressions of FADD were validated in 16 paired HCC tissues of Guangxi cohort. Based on PANoptosis-related genes, an integrated risk signature was constructed to provide a roadmap for patient stratification and predict HCC patient's prognosis. The patients with the higher PANRG-score may carry a dismal survival and relatively low immune infiltration, but a potential better immunotherapy response. Therefore, future HCC therapy perspectives should emphasize the setting of PANoptosis to achieve a personalized, practicable and effective therapeutic regimen.

Identification of NOX4 as a New Biomarker in Hepatocellular Carcinoma and Its Effect on Sorafenib Therapy.

To improve the survival of patients with hepatocellular carcinoma (HCC), new biomarkers and therapeutic targets are urgently needed. In this study, the GEO and TCGA dataset were used to explore the differential co-expressed genes and their prognostic correlation between HCC and normal samples. The mRNA levels of these genes were validated by qRT-PCR in 20 paired fresh HCC samples. The results demonstrated that the eight-gene model was effective in predicting the prognosis of HCC patients in the validation cohorts. Based on qRT-PCR results, NOX4 was selected to further explore biological functions within the model and 150 cases of paraffin-embedded HCC tissues were scored for NOX4 immunohistochemical staining. We found that the NOX4 expression was significantly upregulated in HCC and was associated with poor survival. In terms of function, the knockdown of NOX4 markedly inhibited the progression of HCC in vivo and in vitro. Mechanistic studies suggested that NOX4 promotes HCC progression through the activation of the epithelial-mesenchymal transition. In addition, the sensitivity of HCC cells to sorafenib treatment was obviously decreased after NOX4 overexpression. Taken together, this study reveals NOX4 as a potential therapeutic target for HCC and a biomarker for predicting the sorafenib treatment response.

TRIM55 Promotes Proliferation of Hepatocellular Carcinoma Through Stabilizing TRIP6 to Activate Wnt/β-Catenin Signaling.

Tripartite motif containing 55 (TRIM55) is a member of the TRIM family and functions as an E3 ubiquitin ligase. It acts as a cancer promoter or suppressor in the malignant processes of multiple cancers. However, its proliferative function in hepatocellular carcinoma (HCC) has been poorly studied, and its underlying molecular mechanism remains unclear. In the present study, we investigated the role of TRIM55 in HCC and its mechanism of promoting HCC proliferation. Protein expression levels of TRIM55 were measured in paired HCC and normal tissue samples using immunohistochemical (IHC) staining. The correlation between TRIM55 and clinical features was evaluated by statistical analysis. At the same time, overexpression and knockdown experiments, cycloheximide (CHX) interference experiments, ubiquitination, co-immunoprecipitation and immunofluorescence staining experiments, as well as animal experiments were used to evaluate the potential mechanism that TRIM55 promotes proliferation of hepatocellular carcinoma in vitro and in vivo. TRIM55 expression in HCC specimens was higher compared with the corresponding non-tumor tissues. The overall survival and disease-free survival time of patients with high TRIM55 expression were shorter than those with low expression of TRIM55. Functionally, TRIM55 promoted the proliferation of HCC cells and accelerated the growth of HCC xenografts. Mechanistically, TRIM55 interacted with thyroid receptor interacting protein 6 (TRIP6) and regulate its stability by influencing the ubiquitination process, thereby affecting the Wnt signaling pathway. Our results indicate that TRIM55 promotes HCC proliferation by activating Wnt signaling pathways by stabilizing TRIP6. Therefore, targeting TRIM55 may be an effective therapeutic strategy to inhibit HCC growth.

LncASAP1-IT1 promotes hepatocellular carcinoma progression through the regulation of the miR-1294/TGFBR1 pathway in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the most common tumor with severe morbidity and high mortality. The lncRNA ASAP1-IT1 [the intronic transcript 1 (IT-1) of ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1)] have been shown to promote tumor formation in a variety of cancers. This study sought to investigate the effects of dysregulated ASAP1-IT1 on the biological processes of HCC. The expression levels of ASAP1-IT1 in 30 paired HCC and adjacent non-tumor tissues were measured by real-time-quantitative polymerase chain reaction (RT-qPCR). Several functional tests were performed to investigate the molecular mechanism of ASAP1-IT1 in HCC progression. Our study showed that ASAP1-IT1 was highly expressed in the HCC tissues and cell lines. The knockdown of ASAP1-IT1 inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progression and enhanced the sorafenib sensitivity of the HCC cells. Further investigations revealed that ASAP1-IT1 served as a sponge of microRNA-1294 (miR-1294) to promote transforming growth factor beta receptor 1 (TGFBR1) expression. In addition, the tumor-promoting effect of ASAP1-IT1 was blocked by inhibiting miR-1294/TGFBR1. Tumorigenic assays in nude mice demonstrated that the inhibition of ASAP1-IT1 inhibited the growth of HCC in vivo. These results suggest that lncASAP1-IT1 promotes HCC development by targeting TGFBR1 through miR-1294, which provides a potential target for HCC diagnosis and treatment.

Prognostic role of ring finger and WD repeat domain 3 and immune cell infiltration in hepatocellular carcinoma.

We have found that the expression of ring finger and WD repeat domain 3 (RFWD3) is significantly higher in unpaired and paired hepatocellular carcinoma (HCC) tissues than in normal tissues. Moreover, this expression has a significant correlation with the infiltration level of 14 immune cell types and when the detected RFWD3 expression levels were grouped as high and low, a prominent difference was revealed for overall survival, disease-specific survival, and progression-free interval. Through statistical analysis (univariate Cox), we were also able to identify RFWD3 as an independent prognostic element for HCC, with RFWD3 having an ability to accurately predict HCC prognosis (area under the curve of 0.863). Finally, we have generated prognostic nomograms for probabilities of 1-, 3- and 5-year overall survival in HCC via integrating the factors of age, pathologic stage, alpha-fetoprotein level, and RFWD3 expression.

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.

CircTMEM181 upregulates ARHGAP29 to inhibit hepatocellular carcinoma migration and invasion by sponging miR-519a-5p.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs and are conserved in various species. Although numerous circRNAs have been identified, their role in cancer remains unclear. The expression of circTMEM181 in 90 paired human hepatocellular carcinoma (HCC) and adjacent nontumor tissues were detected using quantitativereverse transcription-polymerase chain reaction. Transwell assay was performed for functional analysis of HCC cell migration and invasion. Luciferase reporter assay was used to verify the combination of circTMEM181 and miR-519a-5p. In this study, we identified a novel circRNA, named circTMEM181, was downregulated in HCC tissues. Decreased expression of circTMEM181 was associated with shorter overall survival of patients with HCC. CircTMEM181 overexpression reduced HCC cell migration and invasion abilities, while circTMEM181 knockdown increased cell motility. Mechanically, circTMEM181 could directly bind to miR-519a-5p and subsequently upregulate ARHGAP29 protein expression. These data provide the first evidence of clinical significance and function of circTMEM181, and suggest the circTMEM181/miR-519a-5p/ARHGAP29 axis in HCC development.

Clinical Significance of TUBGCP4 Expression in Hepatocellular Carcinoma.

We aim to investigate the expression and clinical significance of the tubulin gamma complex-associated protein 4 (TUBGCP4) in hepatocellular carcinoma (HCC). The mRNA expression of TUBGCP4 in HCC tissues was analyzed using The Cancer Genome Atlas (TCGA) database. Paired HCC and adjacent nontumor tissues were obtained from HCC patients to measure the protein expression of TUBGCP4 by immunohistochemistry (IHC) and to analyze the relationship between TUBGCP4 protein expression and the clinicopathological characteristics and the prognosis of HCC patients. We found that TUBGCP4 mRNA expression was upregulated in HCC tissues from TCGA database. IHC analysis showed that TUBGCP4 was positively expressed in 61.25% (49/80) of HCC tissues and 77.5% (62/80) of adjacent nontumor tissues. The Chi-square analysis indicated that the positive rate of TUBGCP4 expression between HCC tissues and the adjacent nontumor tissues was statistically different (P < 0.05). Furthermore, we found that TUBGCP4 protein expression was correlated with carbohydrate antigen (CA-199) levels of HCC patients (P < 0.05). Further, survival analysis showed that the overall survival time and tumor-free survival time in the TUBGCP4 positive group were significantly higher than those of the negative group (P < 0.05), indicating that the positive expression of TUBGCP4 was related to a better prognosis of HCC patients. COX model showed that TUBGCP4 was an independent prognostic factor for HCC patients. Our study indicates that TUBGCP4 protein expression is downregulated in HCC tissues and has a relationship with the prognosis of HCC patients.

Tumor-suppressive role of microfibrillar associated protein 4 and its clinical significance as prognostic factor and diagnostic biomarker in hepatocellular carcinoma.

Revealing microfibrillar-associated protein 4 (MFAP4)'s function and its clinical significance in hepatocellular carcinoma (HCC). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to evaluate MFAP4 mRNA and protein expression in paired HCC and paracarcinoma tissues, respectively. MFAP4 serum concentration was detected using enzyme-linked immunosorbent assays in healthy people (n = 30), cirrhosis (n = 15) and HCC patients (n = 80). MFAP4 protein expression was detected in two tissue microarrays (n = 60 and n = 90). Plasmids were transfected into human HCC cell line Bel-7402, and MFAP4 function was determined in vitro in cell experiments. Furthermore, tumorigenicity studies in nude mice served to assess the function of MFAP4 for HCC. Both MFAP4 mRNA and protein expression were significantly downregulated in HCC tissue compared with paracarcinoma tissue (P < 0.05). Decreased MFAP4 expression in paracarcinoma tissue was associated with poor postoperative survival in HCC patients (P = 0.027). MFAP4 was also downregulated in HCC sera compared with healthy people (P < 0.05). In vitro, MFAP4 upregulation in Bel-7402 cells induced S phase arrest, promoted apoptosis, and inhibited migration and invasion. Western blotting indicated MFAP4 overexpression increased CDK4, CDK6, pRB, P27, and BCL-XS expression. Tumorigenicity study showed that the upregulation of MFAP4 inhibited the proliferation of Bel-7402 cells in nude mice. MFAP4 expression was significantly lower both in sera and tissue of HCC patients. MFAP4 can serve as molecular marker for HCC diagnosis and prognosis. Additionally, MFAP4 acted as an important HCC tumor suppressor by inducing S phase arrest, and promoting apoptosis, cell migration, and invasion.

Tumor-associated macrophages promote resistance of hepatocellular carcinoma cells against sorafenib by activating CXCR2 signaling

BackgroundSorafenib (SOR) is the first line treatment for advanced hepatocellular carcinoma (HCC), but resistance develops frequently. Tumor-associated macrophages (TAMs) have been reported to affect the progression of HCC. We therefore aimed to study the role of TAMs in promoting SOR resistance.MethodsImmunofluorescence staining for the M2 marker CD204 and the cancer stem cell (CSC) markers CD44 and CD133 was performed in paired HCC and adjacent noncancerous tissues and HCC tissues stratified by response of SOR treatment. HCC/U937 coculture system and cytokines were used to induce M2 polarization for studying the effects of M2 TAMs on CSC properties and apoptotic death of HCC cells after SOR treatment.ResultsHigher expression of CD204, CD44, and CD133 was observed in patients with SOR nonresponse (SNR) than in those with SOR response (SR), suggesting that SNR is positively correlated to levels of CSCs and M2 TAMs. After coculture, M2 TAMs could increase the level of CSCs but decrease SOR-induced apoptosis. Incubation of HCC cells with coculture conditioned medium increased the formation of spheres that were resistant to SOR. Furthermore, CXCL1 and CXCL2 were found to be the potential paracrine factors released by M2 TAMs to upregulate SOR resistance in HCC cells. Treatment with CXCL1 and CXCL2 could increase HCC CSC activity but decrease SOR-induced apoptosis by affecting BCL-2 family gene expression. Using pharmacological inhibitors, CXCR2/ERK signaling was found to be critical to CXCL1- and CXCL2-mediated SOR resistance.ConclusionThis study identified CXCL1, CXCL2, and their downstream CXCR2/ERK signaling as potential therapeutic targets to overcome SOR resistance in HCC.

CircRanGAP1/miR-27b-3p/NRAS Axis may promote the progression of hepatocellular Carcinoma

BackgroundThough circular RNAs (circRNAs) are the key regulators in tumor carcinogenesis, they remain largely unexplored in hepatocellular carcinoma (HCC).MethodsThe expression of RanGAP1-derived circRNAs (circ_0063531, circ_0063534, circ_0063513, circ_0063518, circ_0063507, circ_0063723) were evaluated in eight paired HCC and normal tissues, and the correlation between circRanGAP1 (circ_0063531) expression and clinicopathological characteristics in 40 HCC patients was determined. The association between miR-27b-3p and circRanGAP1 or NRAS was predicted using bioinformatics analysis. The expression of circRanGAP1, miR-27b-3p, and NRAS were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The potential oncogenic role of circ-RanGAP1 was assessed using CCK-8, colony formation, transwell assays in vitro, subcutaneous tumor mouse model, vein tail metastatic model, and orthotopically implanted intrahepatic HCC model in vivo. Luciferase reporter and RNA immunoprecipitation (RIP) assays were used to explore the binding site between miR-27b-3p and circ-RanGAP1 or NRAS. Protein expression was detected using western blotting. The localization of miR-27b-3p and circ-RanGAP1 was investigated using fluorescence in situ hybridization (FISH). The level of immune infiltration was assessed by bioinformatics analysis, flow cytometry, and orthotopically implanted intrahepatic HCC models.ResultsHere, we found elevated circRanGAP1 in the cells and clinical tissues of patients with HCC. Increased circRanGAP1 levels are associated with enlarged tumors and the advanced stage of TNM. CircRanGAP1 promotes the growth, migration, and HCC cell invasion, concurrently with the growth and metastasis of tumors in-vivo. Moreover, circRanGAP1 is mainly located inside the cytoplasm. Mechanistically, circRanGAP1 as an oncogene promotes HCC progression by miR-27b-3p/NRAS/ERK axis, furthermore, affects the infiltration level of tumor-associated macrophages probably by sponging miR-27b-3p. Immune infiltration analysis shows that NRAS is positively correlated with the levels of CD68+ tumor-associated macrophages in HCC samples and that NRAS and CD68 are related to the poor outcome of HCC.ConclusionThese results reveal that circRanGAP1 is a HCC oncogene that function by the miR-27b-3p/NRAS/ERK axis and regulates the infiltration levels of tumor-associated macrophages by sponging miR-27b-3p. Therefore, circRANGAP1/ NRAS axis may be an important potential treatment target against HCC.

Prognostic role of expression of angiogenesis markers in hepatocellular carcinoma: A bioinformatics analysis.

The expression of angiopoietin (ANGPT) 1, ANGPT2, vascular endothelial growth factor (VEGF) A, VEGFB, VEGFC, VEGFD, and placental growth factor (PGF) is significantly higher in tumor tissues than in normal tissues in both unpaired and paired hepatocellular carcinoma (HCC) samples. ANGPT2, VEGFB, VEGFC, and PGF are primarily involved in regulating the activation of the epithelial-mesenchymal transition pathway; ANGPT1 is primarily involved in regulating the activation of the RAS/mitogen-activated protein kinase and receptor tyrosine kinase (RTK) pathways; VEGFA is engaged in regulating the RTK activation pathway; and VEGFD is mainly involved in regulating the activation of the tuberous sclerosis protein/mammalian target of rapamycin pathway. There is a significant difference in overall survival between HCC patients with high and low expression of ANGPT2, PGF, VEGFA, and VEGFD. Disease free survival (DFS) is significantly shorter in HCC patients with high ANGPT2, PGF, and VEGFA expression than in those with low ANGPT2, PGF, and VEGFA expression.