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The carcinogenesis and progression of hepatocellular carcinoma (HCC) are closely related to viral infection and intestinal bacteria. However, little is known about bacteria within the HCC tumor microenvironment. Here, we showed that intratumoral Mycoplasma hyorhinis (M. hyorhinis) promoted the initiation and progression of HCC by enhancing nuclear ploidy. We quantified M. hyorhinis in clinical tissue specimens of HCC and observed that patients with high M. hyorhinis load had poor prognosis. We found that gastrointestinal M. hyorhinis can retrogradely infect the liver through the oral-duodenal-hepatopancreatic ampulla route. We further found that the increases in mononuclear polyploidy and cancer stemness resulted from mitochondrial fission caused by intracellular M. hyorhinis. Mechanistically, M. hyorhinis infection promoted the decay of mitochondrial fusion protein (MFN) 1 mRNA in an m6A-dependent manner. Our findings indicated that M. hyorhinis infection promoted pathological polyploidization and suggested that Mycoplasma clearance with antibiotics or regulating mitochondrial dynamics might have the potential for HCC therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

MVI is a risk assessment factor related to hepatocellular carcinoma (HCC) recurrence after hepatectomy or liver transplantation. The goal of this paper is to study the preoperative diagnosis of microvascular invasion (MVI) by using a deep learning algorithm in non-contrast T2 weighted magnetic resonance imaging (MRI) images instead of pathological images. Herein, an ensemble learning algorithm named H-DARnet-based on the difference degree and attention mechanism, combined with radiomics, for MVI prediction-is proposed. Our hybrid network combines the fine-grained, high-level semantic, and radiomics features and exhibits a rich multilevel-feature architecture composed of global-local-prior knowledge with suitable complementarity. The total loss function comprises two regularization items--the triplet and the cross-entropy loss function--which are selected for the triplet network and SE-DenseNet, respectively. The hard triplet sample selection strategy for a triplet network and data augmentation for small-scale liver image datasets in convolutional neural network (CNN) training is indispensable. For 200 patch level test samples (135 positive samples and 65 negative samples), our method can obtain the best prediction results, the AUC, sensitivity, and specificity were 0.826, 79.5% and 73.8%, respectively. The experiment results show that MVI can be predicted by using MRI images, and the proposed method is better than other deep learning algorithms and hand-crafted feature algorithms. The proposed ensemble learning algorithm is proved to be an effective method for MVI prediction., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hydrazinocurcumin (HZC), a synthetic derivative of curcumin (CUR), has been documented to show anticancer potential in impeding tumor growth in several cancers, including hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms remain unclear. This study aimed to explore the function and underlying mechanisms of HZC on HCC cells, which may involve the p38 mitogen activated protein kinase (MAPK) pathway. HZC was first purified and identified. HepG2 cells were then subjected to treatment with HZC or CUR of different concentrations and p38 MAPK signaling inhibitor (SB203580) to verify their effects on HCC cell apoptosis and proliferation. Furthermore, the functional relevance between HZC and the p38 MAPK pathway in HCC was examined. It was observed that 40 μM HZC exhibited the best pro-apoptosis effect in HCC cells. HZC was found to inhibit HCC cell proliferation and promote apoptosis, the effect of which was stronger than 5-fluorouracil (5-FU). More importantly, the anti-oncogenic effect of HZC and 5-FU was implicated with activation of the p38 MAPK pathway. In vivo experimental results showed that HZC inhibited tumor growth more effectively than 5-FU through the p38 MAPK pathway. These results provide evidence that HZC exerted anti-oncogenic and pro-apoptosis effects in HCC cells through activation of the p38 MAPK pathway., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Vasculogenic mimicry (VM) formed by aggressive tumor cells to mimic vasculogenic networks plays an important role in the tumor malignancy of HCC. However, the pathogenesis underlying VM is complex and has not been fully defined. m6A is a common mRNA modification and has many biological effects. However, the relationship between m6A and VM remains unclear. In this research, we found that m6A methyltransferase METTL3 in HCC tissues was positively correlated with VM. The m6A level of mRNA significantly increased in 3D cultured cells treated with VEGFa and was related to VM formation. Transcriptome sequencing analysis of 3D cultured cells with knockdown Mettl3 showed that the Hippo pathway was involved in m6A-mediated VM formation. Further mechanism research indicated that the m6A modification of YAP1 mRNA affected the translation of YAP1 mRNA. In conclusion, m6A methylation plays a key role in VM formation in HCC. METTL3 and YAP1 could be potential therapeutic targets via impairing VM formation in anti-metastatic strategies.

Background: Polymorphism of methylene tetrahydrofolate reductase (MTHFR) C677T has been reported to be associated with HBV-related hepatocellular carcinoma (HCC) risk. However, the underlying mechanism remains elusive. DNA methylation has been suggested to be associated with HCC onset. MTHFR is the key enzyme in folic acid metabolism, thus, it influences the production of the main donor of methyl groups for DNA methylation. This study aimed to determine the association of global DNA methylation with MTHFR C677T polymorphism in chronic HBV infected patients, as well as its association with HCC and gender., Methods: In all, 130 chronic hepatitis B (CHB) and 131 HBV-related HCC patients were enrolled in the study. The methylation level of long interspersed nuclear element-1 (LINE-1), the surrogate marker of global DNA methylation, and MTHFR C677T genotypes were determined., Results: The HCC group showed significantly lower LINE-1 methylation than the CHB group (p = 0.016). Females were observed to have a markedly lower LINE-1 methylation level than males in both CHB and HCC groups (p = 0.000, p = 0.014, respectively). A significant relationship between MTHFR C677T polymorphism and LINE-1 methylation was observed in the CHB group (F = 5.985, p = 0.003). CT, TT, and CT + TT genotypes were significantly associated with lower LINE-1 methylation level compared with the CC genotype (p = 0.005, p = 0.018, p = 0.001, respectively). In addition, the association between MTHFR C677T and LINE-1 methylation was more significant in females (F= 5.036, p = 0.011) than in males (F = 3.083, p = 0.051); further, the association was significant in the subjects older than 60 years (F = 3.865, p = 0.028), but not in the subgroup aged less than 60 years (F = 2.496, p = 0.089)., Conclusions: LINE-1 methylation level in chronic HBV infected patients was associated with the occurrence of HBV-related HCC, gender, and MTHFR C677T polymorphism.

The expression of lysine methyltransferase SET8, which is involved in carcinogenesis of many types of human cancers through monomethylation of histone H4 lysine 20 (H4K20), is associated with the prognosis of hepatocellular carcinoma (HCC). We performed a functional analysis for SET8 to assess its effect on HCC progression. SET8 knockdown inhibited proliferation, migration and invasion of HCC cells. SET8 knockdown also inhibited tumour growth in a human xenograft mouse model. Overexpression of SET8 displayed the reverse effect, while treatment with the SET8 inhibitor UNC0379 produced an effect similar to SET8 knockdown. In addition, drug sensitivity testing in SET8-siRNA transfected HCC cells indicated that docetaxel inhibited cell growth dramatically, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Furthermore, gene expression microarray analysis showed that genes altered after SET8 knockdown were clustered in pathways related to tumorigenesis and metastasis. Our data suggests that targeting SET8 for HCC therapy can inhibit the proliferation and invasion of HCC cells as well as increase their sensitivity to chemotherapy.

Rationale: The role of SLUG in epithelial-mesenchymal transition during tumor progression has been thoroughly studied, but its precise regulation remains poorly explored. Methods: The affinity purification, mass spectrometry and CO-IP were performed to identify the interaction between SLUG and ubiquitin-specific protease 5 (USP5). Cycloheximide chase assays and deubiquitination assays confirmed that the effect of USP5 on the deubiquitin of SLUG. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional regulation of E-cadherin by SLUG effected by USP5. EMT related markers was detected by western blotting and immunofluorescence. Molecular docking, SPR sensor (biacore) and co-location were detected to prove Formononetin targets USP5. Bioinformatics analysis was used to study the relation of USP5 and SLUG to malignancy degree of HCC. Cell migration, invasion in HCC cells and xenografts model in nude mouse were conducted to detect the promotion of USP5 and the inhibition of Formononetin on EMT. Results: USP5 interacts with and stabilizes SLUG to regulate its abundance through USP5 deubiquitination activities in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). USP5 is highly expressed and positively correlated with SLUG expression in HCC with high malignancy. Knockdown of USP5 inhibits SLUG deubiquitination and inhibits HCC cells proliferation, metastasis, and invasion, while overexpression of USP5 promotes SLUG stability and EMT in vitro and in vivo. Through virtual screening, we found that Formononetin exhibits excellent binding to USP5. Moreover, Formononetin inhibits deubiquitinating activities of USP5 to SLUG and consequently impedes the EMT and malignant progression of HCC. Conclusion: Our findings reveal that USP5 serve as a potential target for tumor intervention and provide a preliminary antitumor therapy for inhibit EMT by targeting USP5 or its interaction with SLUG in HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Genetic polymorphism in the epidermal growth factor (EGF, rs4444903) gene has been demonstrated to be associated with the clinical deterioration in hepatitis C virus (HCV)-related liver cirrhosis (LC) and the development of hepatocellular carcinoma (HCC). Whether this single nucleotide polymorphism (SNP) influences susceptibility to HCV-related LC and HCC in the Chinese Han population is largely unknown., Methods: In this case-control study, a total of 187 Chinese Han patients with chronic HCV infection were enrolled, including 62 HCV-related LC patients, 46 HCV-related HCC patients, and 79 chronic hepatitis C (CHC) patients without LC and HCC, and the genetic polymorphism was genotyped via a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) assay. The logistical regression analysis was employed to determine the correlation between the genetic polymorphism and risk of HCV-related LC and HCC., Results: The distribution of EGF rs4444903 genotypes and alleles significantly differed between LC patients and CHC subjects (p = 0.045, p = 0.043, respectively). Under the recessive model, the GG genotype was significantly associated with a two-fold risk of HCV-related LC compared to the AA+AG genotype after an adjustment for age, gender, body mass index (BMI), duration of HCV infection, and HCV RNA level (OR = 2.188; 95% CI = 1.072 - 4.465; p = 0.031). Significant association was observed as well between the GG genotype and increased HCV-related HCC risk (OR = 3.104; 95% CI = 1.319 - 7.307; p = 0.010)., Conclusions: The EGF rs4444903 GG genotype is associated with higher susceptibility to HCV-related LC and HCC in the Chinese Han population. Screening of host genetic polymorphisms might be helpful in designing effective and efficient LC and HCC surveillance programs for chronic HCV-infected patients.

Background: Methylene tetrahydrofolate reductase (MTHFR) is the key enzyme of folic acid metabolism and the C677T mutation is associated with decreased enzyme activity. Several studies have shown its regulatory role in carcinogenesis and tumor growth. HBV (hepatitis B virus)-related HCC (hepatocellular carcinoma) is one of the most common liver cancers worldwide. Therefore, the present case-control study aimed to investigate the role of genetic polymorphism of MTHFR C677T in the development and progression of HBV-related HCC in a Chinese population., Methods: Subjects enrolled included 204 HBV-related HCC patients and 211 HBV infected patients without HCC. MTHFR C677T polymorphism was genotyped via a DNA microarray-based assay. The relationship between the MTHFR C677T polymorphism and HBV-related HCC was analyzed., Results: The genotype frequencies of MTHFR C677T were statistically different between the HCC and control groups (p = 0.025). The TT genotype was associated with elevated risk of HBV-related HCC in a Chinese population under different genetic models after an adjustment for age, gender, HBV infection duration, and HCC family history (T vs. C, OR = 1.462, 95% CI: 1.090 - 1.962, p = 0.011; TT vs. CC, OR = 2.151, 95% CI: 1.143 - 4.049, p = 0.018; TT vs. CC+CT, OR = 1.918, 95% CI: 1.215 - 3.026, p = 0.005). When stratified with the known duration of HBV infection, subjects with HBV infection duration of more than 20 years and carrying the homozygous TT genotype had a higher susceptibility to HCC than those with the C allele (CC/CT) (OR = 2.568, 95% CI: 1.244 - 5.303; p = 0.011). There was no significant association between MTHFR C677T genotypes and HCC stages based on BCLC staging system., Conclusions: MTHFR C677T polymorphism with TT genotype could be a factor that increases the risk of HBVrelated HCC in a Chinese population, especially those with HBV infection duration of more than 20 years.

Background: Vascular endothelial growth factor receptors (VEGFRs) are the major receptors involved in endothelial cell-dependent tumor angiogenesis. There are studies account for the effects of Hsp90 on angiogenesis, but the role and mechanism of Hsp90β isoforms and NVP-BEP800, a specific inhibitor of Hsp90β, in tumor angiogenesis is rarely mentioned., Methods: Immunohistochemistry and statistical analysis was used to evaluate the correlation between Hsp90β expression, CD31 endothelial cell-dependent vessel density, and VEGFRs expression in tissue samples of 96 HCCs. Kaplan-Meier survival analysis and COX proportional hazards analysis the relation of Hsp90β and prognosis. HUVEC cells were transfected with Hsp90β or treated with NVP-BEP800, and then cell proliferation, migration, invasion and tube formation were investigated. The VEGFR1 and VEGFR2 expression was determined by Western blot and immunofluorescence. The VEGFR1 and VEGFR2 promoter activities were detected by dual luciferase report system. In vivo, the angiogenesis promotion of Hsp90β and anti-angiogenesis efficacy of NVP-BEP800 was tested in HCC xenograft models. Histological analysis was performed on tumor samples to evaluate Hsp90β, VEGFRs expression and MVD., Results: This study investigated the correlation between Hsp90β expression and CD31+ endothelial cell-dependent vessel density. Hsp90β promoted VEGFRs expression by increasing their promoter activities. The proliferation, migration, invasion, and tube formation activities of human endothelial cells significantly increased when Hsp90β was overexpressed. NVP-BEP800 down-regulated VEGFRs expression to significantly reduce tubular differentiation, as well as endothelial cell proliferation, migration, and invasion. Furthermore, NVP-BEP800 decreased VEGFR1 and VEGFR2 promoter activities. In vivo, Hsp90β promoted VEGFRs and CD31 expression in human hepatocellular carcinoma tumor xenografts and was associated with increased tumor microvessel density. After 18 days of treatment with 30 mg/kg/day NVP-BEP800, VEGFRs and CD31 expression significantly decreased., Conclusion: Hsp90β induced endothelial cell-dependent tumor angiogenesis by activating VEGFRs transcription. NVP-BEP800 has potential as a therapeutic strategy for inhibiting tumor angiogenesis by decreasing endothelial cell progression and metastasis. It can help develop a therapeutic strategy for tumor treatment through the inhibition of endothelial cell progression and metastasis.

Unlabelled: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death. However, the underlying mechanism during hepatocarcinogenesis remains unclarified. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative strategy for proteome-wide discovery of novel biomarkers in cancers. Hippocalcin-like 1 (HPCAL1) is a calcium sensor protein. However, the biological function of HPCAL1 is poorly understood in cancers, including HCC. Herein, HPCAL1 was identified by SILAC as a novel hepatocarcinogenesis suppressor down-regulated in HCC cell lines and tissues. Importantly, lost expression of HPCAL1 was associated with worse prognosis of HCC patients. Interestingly, secreted HPCAL1 protein in the plasma dropped dramatically in HCC patients compared with healthy donors. Receiver operating characteristic curve analysis showed that serum HPCAL1 at a concentration of 8.654 ng/mL could better predict HCC. Furthermore, ectopic expression of HPCAL1 suppresses cell proliferation, while depletion of HPCAL1 led to increased cell growth both in vitro and in vivo. Mechanistically, HPCAL1 directly interacted with p21(Waf/Cip1) in the nucleus, which requires the EF-hand 4 motif of HPCAL1 and the Cy1 domain of p21. This interaction stabilized p21(Waf/Cip1) in an extracellular signal-regulated kinase 1/2-mitogen-activated protein kinase-dependent manner, which subsequently prevented p21(Waf/Cip1) proteasomal degradation by disrupting SCF(Skp2) and CRL4(Cdt2) E3 ligase complexes, resulting in increased protein stability and inhibitory effect of p21(Waf/Cip1). Notably, the tumor suppressive function of HPCAL1 was dependent on p21 in vitro and in vivo. Consistent with this observation, expression of HPCAL1 and p21(Waf/Cip1) was positively correlated in HCC tissues., Conclusion: These findings highlight a novel tumor suppressor upstream of p21(Waf/Cip1) in attenuating cell cycle progression and provide a promising diagnostic and prognostic factor, as well as a potential therapeutic target for HCC., (© 2015 by the American Association for the Study of Liver Diseases.)

Aberrant activation of the Wnt/β-catenin pathway is frequent in hepatocellular carcinoma (HCC) and contributes to HCC initiation and progression. This abnormal activation may result from somatic mutations in the genes of the Wnt/β-catenin pathway and/or dysregulation of the Wnt/β-catenin pathway. The mechanism for the latter remains poorly understood. Prospero-related homeobox 1 (PROX1) is a downstream target of the Wnt/β-catenin pathway in human colorectal cancer and elevated PROX1 expression promotes malignant progression. However, the Wnt/β-catenin pathway does not regulate PROX1 expression in the liver and HCC cells. Here we report that PROX1 promotes HCC cell proliferation in vitro and tumor growth in HCC xenograft mice. PROX1 and β-catenin levels are positively correlated in tumor tissues as well as in cultured HCC cells. PROX1 can upregulate β-catenin transcription by stimulating the β-catenin promoter and enhance the nuclear translocation of β-catenin in HCC cells, which leads to the activation of the Wnt/β-catenin pathway. Moreover, we show that increase in PROX1 expression renders HCC cells more resistant to sorafenib treatment, which is the standard therapy for advanced HCC. Overall, we have pinpointed PROX1 as a critical factor activating the Wnt/β-catenin pathway in HCC, which promotes HCC proliferation and sorafenib resistance.