Hepatocellular Carcinoma Patient Tissues Research Articles

The Prognostic Significance of CTSV Expression in Patients with Hepatocellular Carcinoma.

Cathepsin V (CTSV) is a cysteine protease peptidase, which is typically upregulated in cancer and is associated with various oncogenic processes, such as angiogenesis, proliferation, apoptosis, and invasion. The study explored the role of CTSV in hepatocellular carcinoma (HCC) and its potential as a potential biomarker. This study collected tumor and peritumoral archived specimens from 180HCC patients who underwent surgical resection at Zhongshan Hospital, Fudan University (Shanghai, China) between 2009 and 2010. We extracted data from the TCGA and GEO databases and conducted differential expression analysis, univariate Cox regression, and Kaplan-Meier analysis. Ultimately, we determined that CTSV may emerge as a potential biomarker. Then, immunohistochemical staining for CTSV was performed on tumors and adjacent tissues of HCC patients, and a Cox proportional hazards model was constructed to evaluate the prognostic significance of CTSV expression levels. Applied functional enrichment analysis to reveal the underlying molecular mechanisms. Utilized ssGSEA enrichment analysis and TIMER2.0 algorithm to explore the correlation between CTSV expression and immune cells in HCC. In vitro and in vivo experiments were conducted using human liver cancer cell lines to further validate the clinical application value of CTSV. In this study, we observed that CTSV expression was notably elevated in HCC (P < 0.001), and identified a significant association between elevated CTSV expression and reduced overall survival rates in patients. In vitro and in vivo experiments indicated that CTSV knockdown could significantly inhibit the proliferation, migration, and invasion of liver cancer cells, and it was found that the combination of CTSV knockdown with PD-1 inhibitors might enhance the therapeutic effect of PD-1 inhibitors in HCC. CTSV serves as a standalone negative prognostic indicator and possesses clinical significance in HCC.

Endosialin-positive CAFs promote hepatocellular carcinoma progression by suppressing CD8+ T cell infiltration

Background and aimsEndosialin, also known as tumor endothelial marker1 or CD248, is a transmembrane glycoprotein that is mainly expressed in cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC). Our previous study...

CircCOCH plays a critical role in Hepatocellular carcinoma through modulating miR-450a and activating PI3K/mTOR pathway

Hepatocellular carcinoma (HCC) is a primary liver cancer with high pathogenicity and extremely poor prognosis. The role of circular RNAs (circRNAs) in HCC carcinogenesis and progression remains to be determined. Based on the analysis of HCC-related databases, as well as the expression analysis and identification of 25 HCC patient tissues and HCC cell lines, we found that the hsa_circ_0031431 (circCOCH) is significantly highly expressed in HCC tissues and cell lines. High circCOCH expression is associated with enhanced tumor proliferation and metastasis, and knocking down circCOCH can inhibit the growth of HCC in vivo and in vitro. Mechanistic studies show that circCOCH upregulates the expression of epidermal growth factor receptor (EGFR) through sponge miR-450a, thereby activating the Phosphoinositide 3-kinases (PI3Ks) cell pathway to promote HCC proliferation and metastasis. Futhermore, we found that IGF2BP3 mediates the biogenesis of circCOCH. The present study provides innovative insights into the role of circRNAs in the etiology of HCC carcinogenesis and might serve as a new promising therapeutic target for HCC.

Expression and clinical significance of NLRC5 in hepatocellular carcinoma

ABSTRACT NLRC5, the largest member of the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family, has been reported to participate in the regulation of immune function and is associated with chronic inflammatory diseases. However, the biological function of NLRC5 in hepatocellular carcinoma (HCC) has not been fully demonstrated. The aim of this study is to evaluate NLRC5 expression in the tumor tissues of HCC patients undergoing surgical treatment, assess its prognostic value, and explore its relationship with critical immune-related molecules within the tumor microenvironment. A total of 100 patients with hepatitis B virus-associated HCC receiving surgical treatment were enrolled in the study. Immunohistochemical results were obtained by scoring the intensity of cellular staining and the percentage of positive cells in the tissue sections. The association between NLRC5 expression levels and the main clinicopathological factors was analyzed by Chi-square test method. The prognostic values were analyzed by COX regression model and the Kaplan-Meier survival curve. Receiver operating characteristic (ROC) curve analysis was performed to assess the predictive performance of NLRC5 in postoperative patients with HCC. IHC showed that high expression of NLRC5 was observed in 67% of HCC tissue samples. Chi-square test showed that NLRC5 was a risk factor associated with tumor number, satellite nodule, and envelope invasion. Kaplan-Meier survival curves and COX survival analysis showed that high expression of NLRC5 was significantly associated with decreased overall survival (OS) in HCC patients (HR = 1.79, 95% CI 1.03–3.12, p = .041). However, univariate logistic regression analysis revealed that NLRC5 showed positive relationship with GZMB and CD8α suggesting its role in immune escape of HCC. ROC curve analysis showed that the combination of tumor number, envelope invasion, and NLRC5 expression (area under the curve = 0.824, sensitivity = 77.30%, specificity = 82.4%) can more accurately evaluate the prognosis of HCC patients compared to the combination of only tumor number and envelope invasion (area under the curve = 0.690, sensitivity = 43.9%, specificity = 94.1%).NLRC5 plays a crucial role in progression of HCC and can be considered as a potential prognostic and predictive biomarker. Targeting NLRC5 may provide an attractive therapeutic approach for HCC.

Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log2 fold change ≤−1.5, Padj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3′UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, “DIANA” tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells

BackgroundIncreased level of serum cholic acid (CA) is often accompanied with decreased CYP2E1 expression in hepatocellular carcinoma (HCC) patients. However, the roles of CA and CYP2E1 in hepatocarcinogenesis have not been elucidated. This study aimed to investigate the roles and the underlying mechanisms of CYP2E1 and CA in HCC cell growth.MethodsThe proteomic analysis of liver tumors from DEN-induced male SD rats with CA administration was used to reveal the changes of protein expression in the CA treated group. The growth of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was assessed with immunofluorescence and confocal microscopy. Western blot analysis was used to examine the expression of CYP2E1, mTOR, AKT, p62, and LC3II/I. A xenograft tumor model in nude mice was used to examine the role of CYP2E1 in CA-induced hepatocellular carcinogenesis. The samples from HCC patients were used to evaluate the clinical value of CYP2E1 expression.ResultsCA treatment significantly increased the growth of HCC cells and promoted xenograft tumors accompanied by a decrease of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulated CYP2E1 expression inhibited the growth of HCC cells, blocked autophagic flux, decreased AKT phosphorylation, and increased mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling. Finally, decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and its expression level in tumors was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT).ConclusionsCYP2E1 downregulation contributes to CA-induced HCC development presumably through autophagy regulation. Thus, CYP2E1 may serve as a potential target for HCC drug development.

The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms

Objectives: To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms. Methods: Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT). Results: Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated (r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 (r=-0.167, P=0.044), the level of serum CEA (r=-0.061, P=0.032), and the level of serum ALT(r=-0.147,P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 (r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT (r=0.164, P=0.029). Conclusion: Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

A Novel Epigenetic Strategy to Concurrently Block Immune Checkpoints PD-1/PD-L1 and CD155/TIGIT in Hepatocellular Carcinoma

Tumor microenvironment is an intricate web of stromal and immune cells creating an immune suppressive cordon around the tumor. In hepatocellular carcinoma (HCC), Tumor microenvironment is a formidable barrier towards novel immune therapeutic approaches recently evading the oncology field. In this study, the main aim was to identify the intricate immune evasion tactics mediated by HCC cells and to study the epigenetic modulation of the immune checkpoints; Programmed death-1 (PD-1)/ Programmed death-Ligand 1 (PD-L1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT)/Cluster of Differentiation 155 (CD155) at the tumor-immune synapse. Thus, liver tissues, PBMCs and sera were collected from Hepatitis C Virus (HCV), HCC as well as healthy individuals. Screening was performed to PD-L1/PD-1 and CD155/TIGIT axes in HCC patients. PDL1, CD155, PD-1 and TIGIT were found to be significantly upregulated in liver tissues and peripheral blood mononuclear cells (PBMCs) of HCC patients. An array of long non-coding RNAs (lncRNAs) and microRNAs validated to regulate such immune checkpoints were screened. The lncRNAs; CCAT-1, H19, and MALAT-1 were all significantly upregulated in the sera, PBMCs, and tissues of HCC patients as compared to HCV patients and healthy controls. However, miR-944–5p, miR-105–5p, miR-486–5p, miR-506–5p, and miR-30a-5p were downregulated in the sera and liver tissues of HCC patients. On the tumor cell side, knocking down of lncRNAs—CCAT-1, MALAT-1, or H19—markedly repressed the co-expression of PD-L1 and CD155 and accordingly induced the cytotoxicity of co-cultured primary immune cells. On the immune side, ectopic expression of the under-expressed microRNAs; miR-486–5p, miR-506–5p, and miR-30a-5p significantly decreased the transcript levels of PD-1 in PBMCs with no effect on TIGIT. On the other hand, ectopic expression of miR-944–5p and miR-105–5p in PBMCs dramatically reduced the co-expression of PD-1 and TIGIT. Finally, all studied miRNAs enhanced the cytotoxic effects of PBMCs against Huh7 cells. However, miR-105–5p showed the highest augmentation for PBMCs cytotoxicity against HCC cells. In conclusion, this study highlights a novel co-targeting strategy using miR-105–5p mimics, MALAT-1, CCAT-1 and H19 siRNAs to efficiently hampers the immune checkpoints; PD-L1/PD-1 and CD155/TIGIT immune evasion properties in HCC.

Expression of LC3A, LC3B and p62/SQSTM1 autophagy proteins in hepatocellular carcinoma (HCC) tissues and the predicted microRNAs involved in the autophagy-related pathway.

Autophagy plays multifaceted roles in regulating hepatocellular carcinoma (HCC) and the mechanisms involved are under-explored. Regulatory microRNAs (miRNAs) have been reported to target autophagy proteins but their roles in HCC is not well studied. Using HCC patient tissues, this study aims to investigate the association of autophagy with several clinicopathological parameters as well as identifying the autophagy-related miRNAs and the possible pathways. Autophagy level in the HCC patient-derived cancer and non-cancer tissues was determined by immunohistochemistry (IHC) targeting SQSTM1, LC3A and LC3B proteins. Significance tests of clinicopathological variables were tested using the Fisher's exact or Chi-square tests. Gene and miRNA expression assays were carried out and analyzed using Nanostring platform and software followed by validation of other online bioinformatics tools, namely String and miRabel. Autophagy expression was significantly higher in cancerous tissues compared to adjacent non-cancer tissues. High LC3B expression was associated with advanced tumor histology grade and tumor location. Nanostring gene expression analysis revealed that SQSTM1, PARP1 and ATG9A genes were upregulated in HCC tissues compared to non-cancer tissues while SIRT1 gene was downregulated. These genes are closely related to an autophagy pathway in HCC. Further, using miRabel tool, three downregulated miRNAs (hsa-miR-16b-5p, hsa-miR-34a-5p, and hsa-miR-660-5p) and one upregulated miRNA (hsa-miR-539-5p) were found to closely interact with the abovementioned autophagy-related genes. We then mapped out the possible pathway involving the genes and miRNAs in HCC tissues. We conclude that autophagy events are more active in HCC tissues compared to the adjacent non-cancer tissues. We also reported the possible role of several miRNAs in regulating autophagy-related genes in the autophagy pathway in HCC. This may contribute to the development of potential therapeutic targets for improving HCC therapy. Future investigations are warranted to validate the target genes reported in this study using a larger sample size and more targeted molecular technique.

Poorly Differentiated Hepatocellular Carcinoma Cells Avoid Apoptosis by Interacting with T Cells via CD40–CD40 Ligand Linkage

Hepatocellular carcinoma (HCC) is associated with elevated soluble CD40 levels in our previous study. Here we aimed to investigate CD40's role in liver tumor progression. We examined CD40 levels in HCC patient tissues and various HCC cell lines, studying their interaction with CD4+T cells. RNA sequencing analysis was performed to explore the mechanisms of CD40 induction. Poorly differentiated HCC tumour tissues exhibited high membrane-bound CD40 (mCD40) expression, in contrast to non-tumour areas. Poorly differentiated HCC cell lines showed high expression of mCD40 with low CD40 promoter methylation which were opposite of well-differentiated ones. Solely modulating CD40 expression in HCC cells exerted no direct consequences on cell growth or appearance. Interestingly, HLFs co-cultured with activated (CD40L+) CD4+ T cells elevated CD40 levels and showed a modest 3.2% of dead cells, then increased to 10.9% underwent pre-neutralizing CD40 condition, while pre-blocking both CD40 and Integrin α5β1 concomitantly caused only 1.9% of cell death. RNA sequencing of co-cultured HLFs with activated CD4+ T cells revealed the upregulation of interferon (IFN) and immune response pathways. Elevated IFN-γ levels in the activated T cell media stimulated the JAK1/STAT3 pathway, resulting in increased CD40 expression in HLF. Collectively, CD40 expression in poorly differentiated HCC cells prevents cell death by interacting with CD40L in activated T cells. Targeting CD40 may represent a promising anticancer therapy.

Abstract 4876: Data-driven discovery of cancer-specific targets for hepatocellular carcinoma using spatial transcriptomics

Abstract Background: Hepatocellular carcinoma (HCC) is frequently diagnosed in advanced stages, with limited options available for systemic treatment. Given the significant investment of time, the high costs, and the notable failure rates involved in developing new drugs, it is crucial to identify appropriate cancer-specific surface targets, especially aiming at antibody drug conjugates and radioligand therapy. This study aims to propose a pipeline utilizing spatial transcriptomics (ST) to prioritize potential cancer-specific surface targets for HCC. Methods: Visium ST samples were collected from fresh-frozen tissues of HCC patients undergoing hepatectomy. The spots with over 500 expressed genes and a mitochondrial gene percentage below 20% were selected. Counts were normalized with sctransform, and the tumor cell proportion in each spot was predicted using CellDART. Cancer region-specific genes were identified based on three criteria: high topological overlap with tumor cell distribution (using STopover), a high correlation coefficient with tumor cell proportion, or significantly higher expression in the tumor cell cluster compared to others in single-cell RNA-sequencing dataset. Genes identified through these methods were pooled and filtered based on average expression in the whole tissue using human normal cell atlas data and surface proteins. To select drug candidates with low normal organ toxcity, Tabula Sapiens datasets were used. Genes expressed in over 50% of cells and with an average expression above 1 in any critical organs were excluded. Results: One ST sample was obtained from each of the 7 HCC patients. The topology, correlation, and single-cell-based criteria resulted in 42, 15, and 7 genes, respectively, with only 1 gene (P4HB) overlapping across all three methods. After filtering genes with high expression in normal organs based on Tabula Sapiens, 25 genes remained. In the GEPIA databases, GPC3 exhibited the highest log fold change in RNA expression between HCC and normal liver (6.280), followed by TM4SF4 (2.156), EFNA1 (0.581), GJB1 (0.498), and IRS1 (0.391). To externally validate the results, 17 ST datasets from 5 patients provided by HCCDB were used. Among the selected genes, only GPC3 and EFNA1 had higher average expression in the tumor compared to other compartments (normal, stromal, and immune) in at least 3 out of 5 patients. Conclusion: The proposed platform prioritizes cancer-specific surface targets for HCC, focusing primarily on spatial gene expression profiles obtained from ST and gene expression data from external databases. This analytical tool can be further utilized to identify molecular targets for developing antibody drug conjugates or radioligand therapies, which are designed to specifically deliver therapeutic agents to the tumor. Citation Format: Sungwoo Bae, Dongjoo Lee, Daeseung Lee, Kwon Joong Na, Suk Kyun Hong, YoungRok Choi, Nam-Joon Yi, Kwang-Woong Lee, Kyung-Suk Suh, Hongyoon Choi. Data-driven discovery of cancer-specific targets for hepatocellular carcinoma using spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4876.

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro.

Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial. To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro. HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively. Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 μg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, P < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, P < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down. PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6.

Tumor-associated macrophages (TAMs) are critical in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC). Major vault protein (MVP) mediates multidrug resistance, cell growth and development, and viral immunity. However, the relationship between MVP and TAMs polarization has not been clarified in HCC. We found that MVP significantly increased M2-TAMs infiltration levels in tumor tissues of HCC patients. MVP promoted HCC proliferation, metastasis, and invasion by regulating M2 polarization in vivo and in vitro. Mechanistically, MVP associated with signal transducer and activator of transcription 6 (STAT6) and enhanced STAT6 phosphorylation. STAT6 translocated from the cytosol to the nucleus and regulated M2 macrophage-associated gene transcription. These findings suggest that MVP modulates the macrophage M2 transcriptional program, revealing its potential role in the TAMs of TME.

Clinicopathologic Significance of Quaking Expression in Hepatocellular Carcinoma.

The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of QKI expression have not yet been fully elucidated. In this study, we aimed to investigate the clinicopathological and prognostic significance of QKI expression in hepatocellular carcinoma (HCC). We performed QKI, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and glutathione peroxidase 4 (GPX4) immunohistochemical staining on 166 HCC patient tissue samples. X-tile bioinformatics software was used to set the cut-off value for high QKI expression. Correlations between QKI expression and various clinicopathological parameters were assessed. The best cut-off value for high QKI expression was 12.5. High QKI expression was observed in 28 of 166 patients (16.9%) and was an independent prognostic factor for inferior recurrence-free survival (RFS). In addition, high ZEB1 and GPX4 expression correlated with high QKI expression, but not with the loss of E-cadherin expression. High QKI expression was identified in HCCs and associated with poor RFS. QKI might be a prognostic biomarker of HCCs associated with epithelial-to-mesenchymal transition and a potential candidate therapeutic target.

Mitochondrial ribosomal protein L12 potentiates hepatocellular carcinoma by regulating mitochondrial biogenesis and metabolic reprogramming

BackgroundMitochondrial dysfunction and metabolic reprogramming are key features of hepatocellular carcinoma (HCC). Despite its significance, the precise underlying mechanism behind these processes has not been fully elucidated. The latest investigations, along with our previous discoveries, have substantiated the significant role of mitochondrial ribosomal protein L12 (MRPL12), a newly identified gene involved in mitochondrial transcription regulation, in the modulation of mitochondrial metabolism. Nevertheless, the role of MRPL12 in tumorigenesis has yet to be investigated. MethodsThe expression of MRPL12 in HCC was assessed using an online database. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) were employed to determine the expression of MRPL12 in HCC tissues, patient-derived organoid (PDO), and cell lines. The correlation between MRPL12 expression and clinicopathological features, as well as prognosis, was examined using tissue microarray analysis. An in vivo subcutaneous tumor xenograft model, gene knockdown or overexpression assay, chromatin immunoprecipitation (ChIP) assay, Seahorse XF96 assay, and cell function assay were employed to investigate the biological function and potential molecular mechanism of MRPL12 in HCC. ResultsA significant upregulation of MRPL12 was observed in HCC cells, PDO and patient tissues, which correlated with advanced tumor stage, higher grade and poor prognosis. MRPL12 overexpression promoted cell proliferation, migration, and invasion in vitro, as well as tumorigenicity in vivo, whereas MRPL12 knockdown showed the opposite effect. MRPL12 knockdown also inhibited the capacity of organoids proliferation capacity. Furthermore, MRPL12 was found to be crucial for maintaining mitochondrial homeostasis. Both gain and loss-of-function experiments targeting MRPL12 in HCC cells altered oxidative phosphorylation (OXPHOS) and mitochondrial DNA content. Notably, suppression of OXPHOS effectively mitigates the tumor-promoting effect attributed to MRPL12 overexpression, implying the involvement of MRPL12 in HCC through the modulation of mitochondrial metabolism. Besides, Yin Yang 1 (YY1) was identified as a transcription factor responsible for regulating MRPL12, while the PI3K/mTOR pathway was found to act as an upstream regulator of YY1. MRPL12 knockdown attenuated the YY1 overexpression or PI3K/mTOR activation-induced malignant phenotype in HCC cells. ConclusionOur findings provide compelling evidence that MRPL12 is implicated in driving the malignant phenotype of HCC via regulating mitochondrial metabolism. Moreover, the aberrant expression of MRPL12 in HCC is mediated by the upstream PI3K/mTOR/YY1 pathway. These results highlight the potential of targeting MRPL12 as a promising therapeutic strategy for the treatment of HCC.

Cyclin A1 affects the invasion, metastasis, and prognosis of hepatocellular carcinoma

Objective: To investigate the effect of cyclin A1 on the invasion, metastasis, and prognosis of hepatocellular carcinoma (HCC). Methods: Immunohistochemistry (IHC) was used to detect the expressional condition of cyclin A1 in HCC and paraffin-embedded non-tumor adjacent tissues. Kaplan-Meier method was used for the survival analysis of patients with HCC. Western blot (WB) was used to detect the expression of cyclin A1 in HCCLM3 and QGY-7703 cells. Scratch wound healing assay, transwell migration, and invasion assay were used to detect the effect of cyclin A1 overexpression on cell migration and invasion ability. WB was used to detect changes in the expression of matrix metalloproteinase (MMP) 2, MMP9, and vascular endothelial growth factor (VEGF) after overexpression of cyclin A1. Measurement data were compared using a t-test and analysis of variance. Count data was measured using χ (2) test and the Log-rank method was performed for survival analysis. Results: Cyclin A1 expression rates were higher in the tissues of HCC patients with recurrent metastasis than in the tissues of patients without recurrent metastasis (60.42% vs. 46.81%, χ (2) = 4.711, P < 0.05). The overall postoperative survival time (OS) and disease-free survival (DFS) were shorter in patients with high cyclin A1 expression than those with low cyclin A1 expression (45.9 months vs. 53.1 months; 42.9 months vs. 51.3 months, and P < 0.01). The postoperative OS and DFS were shorter in patients with high cyclin A1 expression and recurrent metastasis than those with low cyclin A1 expression without recurrent metastasis (31.7 months vs. 43.9 months; 18.0 months vs. 31.5 months, and P < 0.05). HCCLM3 and QGY-7703 cells were higher in the cyclin A1-pEX group than in the empty vector (vector) group (1.56 ± 0.06 vs. 0.18 ± 0.01, t = 18.75, P < 0.001; 1.31 ± 0.05 vs.0.37 ± 0.02, t = 15.17, P < 0.001). The migrated distances of HCCLM3 cells in the cyclin A1-pEX group and the vector group were (536.7 ± 14.5) μm and (327.3 ± 9.3) μm, t = 11.84, P < 0.05, respectively, while the migrated distances of QGY-7703 cells in the two groups were (916.7 ± 35.3) μm and (320.0 ± 20.8) μm, t = 13.54, P < 0.01. The migrated numbers of HCCLM3 cells in the cyclin A1-pEX group and vector group were (37.3 ± 2.4) and (7.0 ± 1.2), t = 12.67, P < 0.001, and the number of invasive cells was (73.7 ± 4.1) and (12.6 ± 1.5), t = 12.36, P < 0.001, respectively. The migrated numbers of QGY-7703 cells in the two groups were (153.3 ± 6.0) and (17.7 ± 3.7), t = 17.59, P < 0.001, and the number of invasive cells was (45.0 ± 2.9) and (9.3 ± 1.5), t = 10.66, P < 0.001, respectively. The expression levels of MMP2, MMP9, and VEGF in HCCLM3 and QGY-7703 cells were significantly higher in the cyclin A1-pEX group than those in the vector group (P < 0.05). Conclusion: Cyclin A1 plays an important role in HCC invasion and metastasis, but HCC patients with high cyclin A1 expression have a poor prognosis. Hence, cyclin A1 has high guiding significance for evaluating patient prognosis.

ADAMTS12 is a stromal modulator in chronic liver disease.

Adamalysins, a family of metalloproteinases containing a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTSs), belong to the matrisome and play important roles in various biological and pathological processes, such as development, immunity and cancer. Using a liver cancer dataset from the International Cancer Genome Consortium, we developed an extensive in silico screening that identified a cluster of adamalysins co-expressed in livers from patients with hepatocellular carcinoma (HCC). Within this cluster, ADAMTS12 expression was highly associated with recurrence risk and poorly differentiated HCC signatures. We showed that ADAMTS12 was expressed in the stromal cells of the tumor and adjacent fibrotic tissues of HCC patients, and more specifically in activated stellate cells. Using a mouse model of carbon tetrachloride-induced liver injury, we showed that Adamts12 was strongly and transiently expressed after a 24 h acute treatment, and that fibrosis was exacerbated in Adamts12-null mice submitted to carbon tetrachloride-induced chronic liver injury. Using the HSC-derived LX-2 cell line, we showed that silencing of ADAMTS12 resulted in profound changes of the gene expression program. In particular, genes previously reported to be induced upon HSC activation, such as PAI-1, were mostly down-regulated following ADAMTS12 knock-down. The phenotype of these cells was changed to a less differentiated state, showing an altered actin network and decreased nuclear spreading. These phenotypic changes, together with the down-regulation of PAI-1, were offset by TGF-β treatment. The present study thus identifies ADAMTS12 as a modulator of HSC differentiation, and a new player in chronic liver disease.

Hsa_circ_0119412 is a tumor promoter in hepatocellular carcinoma by inhibiting miR-526b-5p to upregulate STMN1

ABSTRACT Hepatocellular carcinoma (HCC) is always deemed a deadly malignancy worldwide. Non-coding RNAs, including circRNAs, are becoming more widely recognized as essential regulators of the malignant development of HCC. Thus, we elaborated the regulating role of hsa_circ_0119412 in HCC advancement. The qRT-PCR was done to estimate the expressions of hsa_circ_0119412, miR-526b-5p, and Stathmin 1 (STMN1) in HCC (clinical samples and cell lines), and immunoblotting was used to detect STMN1 protein level in HCC cell lines. The stability of the circRNA was checked by processing with ribonuclease R. The proliferative potential of HCC cells was examined via the CCK-8 assay and the migratory potential by the wound healing assay. Immunoblotting was done to examine Bax and Bcl-2 (apoptosis-related proteins). Luciferase and RIP assays were employed to establish the direct interactions among miR-526b-5p and hsa_circ 0119412/STMN1. In vivo tumor growth was measured by doing a xenograft tumor experiment. In the tissues of HCC patients and cell lines derived from HCC cells, hsa_circ_0119412 was distinctly over-expressed. Knocking down hsa_circ_0119412 impeded proliferation and migration while inducing apoptosis in HCC cells. Moreover, silencing hsa_circ_0119412 diminished tumor weight and volume in vivo. Interestingly, miR-526b-5p inhibition partially restored the anti-tumor effects of silencing hsa_circ_0119412. STMN1 expression was also abundant in HCC, suggesting that it play a tumor-promoting role. Mechanistically, hsa_circ_0119412 sponged miR-526b-5p, resulting in STMN1 upregulation and thus facilitating the progression of HCC. In conclusion, this study reveals that hsa_circ_0119412 knockdown attenuates the progression of HCC by targeting miR-526b-5p/STMN1 axis.

Interplay between SUMO1-related SUMOylation and phosphorylation of p65 promotes hepatocellular carcinoma progression

The nuclear factor kappaB (NF-κB) subunit p65, plays an important role in the progression of hepatocellular carcinoma (HCC). Phosphorylation of p65 is considered as an important mechanism for the positive regulation of NF-κB activity. According to our previous data, p65 can be SUMOylated by small ubiquitin-related modifier 1 (SUMO1) protein, and SUMO1 promotes p65 nuclear import and HCC progression. However, the effect of SUMO1-related p65 SUMOylation on NF-κB transcriptional activity and the relationship between phosphorylation and SUMOylation of p65 remain obscure. Here, we found that phosphorylated p65 level was increased in cancer tissues of HCC patients, and similar phenomenon was found for SUMO1 expression but not for SUMO2/3. Further clinical data showed a positive correlation between SUMO1 and phosphorylated p65. We also verified that overexpression of SUMO1 upregulated phosphorylated p65 levels. Next, we verified SUMO1-related p65 SUMOylation with in vitro SUMOylation assay, constructed mutants of p65 SUMOylation and phosphorylation, and found that SUMO1-related p65 SUMOylation promoted p65 nuclear import and increased NF-κB activity. Both SUMO1-related p65 SUMOylation and p65 phosphorylation (especially at S276 site) increased the viability and invasion of hepatoma cells, and decreased the apoptosis of hepatoma cells. At last, we found that the phosphorylation of p65 promoted the level of SUMO1-related p65 SUMOylation, and SUMO1-related p65 SUMOylation upregulated phosphorylated p65 (at S276 site). Our study contributes to the exploration of the oncogenic mechanism of p65, which is the important protein in HCC.

TRIM14 Overexpression Induces Chemoresistance and Malignant Behaviors of Hepatocellular Carcinoma Cells by Activating the STAT3/HIF-1α Pathway.

Members of the tripartite motif (TRIM)-containing protein family have been found to be involved in the progression of hepatocellular carcinoma (HCC). TRIM14 exerts a promotive impact on several cancers. This study aimed to explore the function and mechanism of TRIM14 in HCC. TRIM14 expression in HCC tissues and HCC cell lines was detected. The overexpression or knockdown model of TRIM14 was established in HCC cell lines. Cell Counting Kit-8 (CCK-8) assay, flow cytometry, Transwell assay, RT-PCR, Western blot, and immunofluorescence were performed to verify the influence of TRIM14 on cell proliferation, sensitivity to chemotherapy drugs, apoptosis, migration, invasion, and autophagy. A xenograft tumor model was used to confirm the impact of TRIM14 on tumor cell growth. As shown by the data, TRIM14 level was notably higher in the tumor tissues of HCC patients than in the adjacent tissues. The overall survival rate of patients with a high TRIM14 expression was relatively lower than that of patients with a low TRIM14 expression. TRIM14 upregulation enhanced the proliferation, autophagy, migration, and invasion of HCC cells and chemoresistant HCC cells and decreased apoptosis. TRIM14 knockdown contributed to the opposite effects. In in vivo experiments, TRIM14 upregulation bolstered tumor growth. Western blot analysis revealed that TRIM14 upregulation boosted signal transducer and activator of transcription3 (STAT3) and hypoxia-inducible factor-1alpha (HIF-1α) expression, and TRIM14 knockdown suppressed their expression. Moreover, repressing STAT3 and HIF-1α could mitigate the tumor-promoting role of TRIM14 in HCC cells. Overall, TRIM14 facilitated malignant HCC development and induced chemoresistance in HCC cells by activating the STAT3/HIF-1α axis.