Hepatocellular Carcinoma Invasion Research Articles

Massive Upper GI Bleeding from Duodenal Invasion of hepatocellular carcinoma successfully treated with embolization and Hemospray®

Massive Upper GI Bleeding from Duodenal Invasion of hepatocellular carcinoma successfully treated with embolization and Hemospray®

HnRNPA2B1 promotes the occurrence and progression of hepatocellular carcinoma by downregulating PCK1 mRNA via a m6A RNA methylation manner

BackgroundN6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.MethodsMultiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.ResultshnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.ConclusionOur study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.

EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A

BackgroundEpigenetic alterations play an important role in hepatocellular carcinoma (HCC) development. Enhancer of zeste homolog 2 (EZH2) is a well-known epigenetic modifier that functions as an oncogene in tumors by promoting the H3K27me3-mediated transcriptional repression of tumor suppressor genes. “Senescent cells” has been proposed as a possible core component of the hallmarks of cancer conceptualization. Induction of cell senescence and targeted elimination of these senescent tumor cells are new strategies for tumor therapy. However, the role of EZH2 in regulating cellular senescence remains poorly understood.MethodsBioinformatics analyses suggested that EZH2 and DNA topoisomerase II alpha (TOP2A) are coexpressed in tumors, including HCC. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses and gene set enrichment analyses (GSEA) suggests a correlation of EZH2 and TOP2A expression with cellular senescence in HCC. MicroRNA (miRNA) inhibitor and mimics, siRNA, PLKO-shRNA, and plenti6.3-miR-139 were used to upregulate or downregulate the expression of target genes. CCK8, EdU, clone formation, and senescence-associated β-galactosidase (SA-β-gal) staining assays were performed to assess cell proliferation and cellular senescence phenotypes. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to investigate the targeted binding and inhibition of TOP2A 3′ untranslated region (UTR) by miR-139-5p and the DNA enrichment of miR139-5p by EZH2 and H3K27me3. BALB/c nude mice were used to establish a xenograft tumor model and verify the phenotypes upon EZH2 and TOP2A silencing and miR-139 overexpression in vivo. In addition, tissue microarrays were used to analyze the expression patterns and correlations among EZH2, TOP2A, and miR-139-5p expression in HCC.ResultsBioinformatics analysis revealed that EZH2 and TOP2A are coexpressed in HCC. In vitro gain- and loss-of-function experiments showed that inhibition of EZH2 and TOP2A induces cellular senescence and inhibits proliferation of HCC cells. In vivo tumorigenesis assays indicated that EZH2 and TOP2A knockdown inhibits tumorigenesis by inducing cellular senescence. Mechanistically, EZH2 promotes TOP2A expression by regulating the H3K27me3-mediated epigenetic silencing of miR-139-5p. TOP2A is a direct target of miR-139-5p, and inhibition of miR-139-5p can reverse the promotion by EZH2 of TOP2A expression. The overexpression of miR-139-5p induces cellular senescence and inhibits proliferation of HCC cells both in vitro and in vivo. Clinically, expression of EZH2 and TOP2A are higher in HCC tissues than in normal tissues, and this high coexpression indicates a worse outcome of patients with HCC. Moreover, expression of EZH2 and TOP2A is significantly correlated with tumor differentiation grade, tumor invasion, and TNM stage in HCC. miR-139-5p expression is lower in HCC tumors than in normal tissues and is correlated with better prognosis of HCC patients.ConclusionsOur study revealed the role of the EZH2/miR-139-5p/TOP2A axis in regulating cellular senescence and cell proliferation in HCC, enriching the molecular mechanisms of EZH2-mediated epigenetic regulation in HCC. Therefore, our results provide insight into the therapeutic potential of targeting EZH2 to induce cellular senescence and then destroy senescent cells for HCC.Graphic

DNA methylation-activated full-length EMX1 facilitates metastasis through EMX1-EGFR-ERK axis in hepatocellular carcinoma

Altered DNA methylation is a crucial epigenetic event in hepatocellular carcinoma (HCC) development and progression. Through methylation-transcriptomic analysis, we identified a set of sixty potential DNA methylation-based epidriver genes. In this set of genes, we focused on the hypermethylation of EMX1, which is frequently observed in hepatobiliary tumors. Despite of its frequent occurrence, the function of EMX1 remains largely unknown. By utilizing bisulfite-next-generation sequencing, we have detected EMX1 DNA hypermethylation on the gene body, which is positively correlated with EMX1 mRNA expression. Further analysis revealed that EMX1 mRNA terminal exon splicing in HCC generated two protein isoforms: EMX1 full length (EMX1-FL) and alternative terminal exon splicing isoform (EMX1-X1). Cellular functional assays demonstrated that gain-of-function EMX1-FL, but not EMX1-X1, induced HCC cells migration and invasion while silencing EMX1-FL inhibited HCC cells motility. This result was further validated by in vivo tumor metastasis models. Mechanistically, EMX1-FL bound to EGFR promoter, promoting EGFR transcription and activating EGFR-ERK signaling to trigger tumor metastasis. Therefore, EGFR may be a potential therapeutic target for EMX1-high expression HCC. Our work illuminated the crucial role of gene body hypermethylation-activated EMX1-FL in promoting tumorigenesis and metastasis in HCC. These findings pave the way for targeting the EMX1-EGFR axis in HCC tumorigenicity and metastasis.

Targeting B4GALT7 suppresses the proliferation, migration and invasion of hepatocellular carcinoma through the Cdc2/CyclinB1 and miR-338-3p/MMP2 pathway.

As a three-dimensional network involving glycosaminoglycans (GAGs), proteoglycans (PGs) and other glycoproteins, the role of extracellular matrix (ECM) in tumorigenesis is well revealed. Abnormal glycosylation in liver cancer is correlated with tumorigenesis and chemoresistance. However, the role of galactosyltransferase in HCC (hepatocellular carcinoma) is largely unknown. Here, the oncogenic functions of B4GALT7 (beta-1,4-galactosyltransferase 7) were identified in HCC by a panel of in vitro experiments, including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), colony formation, transwell and flow cytometry assay. The expression of B4GALT7 in HCC cell lines and tissues were examined by qPCR (real-time quantitative polymerase chain reaction) and western blot assay. The binding between B4GALT7 and miR-338-3p was examined by dual-luciferase reporter assay. B4GALT7 encodes galactosyltransferase I and it is highly expressed in HCC cells and human HCC tissues compared with para-tumor specimens. MiR-338-3p was identified to bind the 3' UTR (untranslated region) of B4GALT7. Highly expressed miR-338-3p suppressed HCC cell invasive abilities and rescued the tumor-promoting effect of B4GALT7 in HCC. ShRNA (short hairpin RNA) mediated B4GALT7 suppression reduced HCC cell invasive abilities, and inhibited the expression of MMP-2 and Erk signaling. These findings identified B4GALT7 as a potential prognostic biomarker and therapeutic target for HCC.

Refining MRI-based criteria for portal vein invasion in hepatocellular carcinoma: improving sensitivity beyond portal vein tumor thrombosis.

To investigate the imaging features indicating portal vein invasion (PVI) of hepatocellular carcinoma (HCC) on gadoxetic acid-enhanced MRI and to create more accurate diagnostic criteria than the presence of portal vein tumor thrombosis (PVTT) on MRI. This retrospective study included patients with surgically resected HCC larger than 5 cm, and the presence of PVI was investigated. On MRI, we evaluated the image findings of portal vein occlusion, the parenchymal signal change caused by hemodynamic alterations of the portal vein, and their combination showing the highest odds ratio (OR) to define the diagnostic criteria for radiological PVI detection (rPVI criteria). The diagnostic performance and recurrence-free survival were compared between the rPVI criteria and the presence of PVTT using McNemar's test and Kaplan-Meier method, respectively. Interobserver agreement was evaluated using Cohen's weighted ĸ statistics. Of 189 enrolled patients, 25 (13.2%) had PVI on histology. To diagnose PVI on MRI, either peripheral wedge-shaped arterial peritumoral hyperemia with an abrupt cut-off of a portal vein or the presence of PVTT had the highest OR (41.67, p<0.001). The sensitivity of PVI was significantly increased under this diagnostic criterion (64.0% to 88.0%; p=0.031) with comparable accuracy (95.2% vs. 94.7%; p>0.999). In terms of recurrence-free survival, the patient group with rPVI was significantly worse (p=0.017) compared with the patients without rPVI. Interobserver agreement of radiologic findings was substantial (ĸ=0.64). Diagnostic criteria for radiologically PVI detection increase the sensitivity more than the only presence of PVTT.

Single-cell dissection of the multicellular ecosystem and molecular features underlying microvascular invasion in HCC.

Microvascular invasion (MVI) is a crucial pathological hallmark of HCC that is closely associated with poor outcomes, early recurrence, and intrahepatic metastasis following surgical resection and transplantation. However, the intricate tumor microenvironment and transcriptional programs underlying MVI in HCC remain poorly understood. We performed single-cell RNA sequencing of 46,789 individual cells from 10 samples of MVI+ (MVI present) and MVI- (MVI absent) patients with HCC. We conducted comprehensive and comparative analyses to characterize cellular and molecular features associated with MVI and validated key findings using external bulk, single-cell, and spatial transcriptomic datasets coupled with multiplex immunofluorescence assays. The comparison identified specific subtypes of immune and stromal cells critical to the formation of the immunosuppressive and pro-metastatic microenvironment in MVI+ tumors, including cycling T cells, lysosomal associated membrane protein 3+ dendritic cells, triggering receptor expressed on myeloid cells 2+ macrophages, myofibroblasts, and arterial i endothelial cells. MVI+ malignant cells are characterized by high proliferation rates, whereas MVI- malignant cells exhibit an inflammatory milieu. Additionally, we identified the midkine-dominated interaction between triggering receptor expressed on myeloid cells 2+ macrophages and malignant cells as a contributor to MVI formation and tumor progression. Notably, we unveiled a spatially co-located multicellular community exerting a dominant role in shaping the immunosuppressive microenvironment of MVI and correlating with unfavorable prognosis. This study provides a comprehensive single-cell atlas of MVI in HCC, shedding light on the complex multicellular ecosystem and molecular features associated with MVI. These findings deepen our understanding of the underlying mechanisms driving MVI and provide valuable insights for improving clinical diagnosis and developing more effective treatment strategies.

AKR1B10 inhibits the proliferation and metastasis of hepatocellular carcinoma cells by regulating the PI3K/AKT pathway.

Hepatocellular carcinoma (HCC) is one of the most frequent and aggressive malignant neoplasms, and is associated with a poor prognosis. Therefore, there is a crucial need to develop novel cancer therapies and identify novel therapeutic targets. Aldo-keto reductase family 1 member B10 (AKR1B10) is expressed in various types of cancer. However, the role of AKR1B10 in the pathological process of HCC and its underlying molecular mechanism is poorly understood. AKR1B10 expression was evaluated pan-cancer and in HCC using the Genomic Data Commons-The Cancer Genome Atlas (GDC-TCGA) and International Cancer Genome Consortium (ICGC) databases. The relationship between elevated AKR1B10 expression and overall survival in HCC patients was analyzed using a Kaplan-Meier plot. The effects of AKR1B10 on the proliferation, migration, and invasion of HCC cells were evaluated. The proliferation of HCC was measured using CCK-8 and colony formation assays. Transwell and wound healing assays were used to assess the migration and invasion of HCC cells. Western blots were used to detect the expression of proliferative and epithelial-mesenchymal transition (EMT) related proteins in HCC cells, including CCND1, E-cadherin, N-cadherin, vimentin, Twist1, PI3K/p-PI3K, and AKT/p-AKT. AKR1B10 expression was significantly upregulated pan-cancer and in liver cancer. Upregulated AKR1B10 expression was associated with a worse overall survival. HCC cell proliferation, migration, and invasion were found to be influenced by AKR1B10 activity, as demonstrated using DepMap analysis. AKR1B10 knockdown in Huh7 cells reduced proliferation, migration, invasion, and EMT. Mechanistically, AKR1B10 increased the expression of proliferative and EMT-related proteins CCND1, E-cadherin, N-cadherin, vimentin, and Twist1. PI3K and AKT phosphorylation levels decreased following AKR1B10 knockdown. In conclusion, AKR1B10 promoted the proliferation, migration, and invasion of HCC cells via the PI3K/AKT signaling pathway, a potential prognostic indicator.

Oxoaporphine Pr(III) complex inhibits hepatocellular carcinoma progression and metastasis by disrupting tumor cell–macrophage crosstalk

Tumor cells and macrophages communicate through the secretion of various cytokines to jointly promote the malignant development of cancers. We synthesized and characterized an oxoaporphine Pr(III) complex (PrL3(NO3)3) and found that it inhibits hepatocellular carcinoma (HCC) progression and metastasis by disrupting HCC cell–macrophage crosstalk. PrL3(NO3)3 treatment upregulated CD86, TNF-α, and IL-1β and downregulated CD163, CD206, CCL2, and VEGFA in macrophages. Our mRNA-Seq results demonstrated that PrL3(NO3)3 inhibited macrophage M2-like polarization by inhibiting the AMPK pathway and activating the NF-κB pathway by upregulating RelA/p65 Ser536 phosphorylation. This kind of macrophage polarization significantly inhibited HCC cell proliferation, migration, and invasion. In addition, PrL3(NO3)3 inhibited the migration, invasion, and chemotaxis of HCC cells by downregulating the expression of EMT-related markers and CCL2. hTFtarget database analysis revealed that PrL3(NO3)3 inhibited NF-κB nuclear translocation by upregulating RelA/p65 Ser536 phosphorylation in HCC cells, thereby downregulating the expression of Snail and CCL2. HCC tissue microarray analysis revealed that downregulation of RelA/p65 Ser536 phosphorylation is a driving event in HCC malignant progression. In conclusion, PrL3(NO3)3 effectively inhibits HCC cell–macrophage crosstalk by upregulating RelA/p65 Ser536 phosphorylation. This is the first report of a lanthanide complex exerting regulatory effects on both tumors and tumor-associated macrophages, providing a new strategy for the development of effective antitumor drugs.

Kidney-type glutaminase is a biomarker for the diagnosis and prognosis of hepatocellular carcinoma: a prospective study

PurposeThe pathological diagnosis and prognosis prediction of hepatocellular carcinoma (HCC) is challenging due to the lack of specific biomarkers. This study aimed to validate the diagnostic and prognostic efficiency of Kidney-type glutaminase (GLS1) for HCC in prospective cohorts with a large sample size.MethodsA total of 1140 HCC patients were enrolled in our prospective clinical trials. Control cases included 114 nontumour tissues. The registered clinical trial (ChiCTR-DDT-14,005,102, chictr.org.cn) was referred to for the exact protocol. GLS1 immunohistochemistry was performed on the whole tumour section. The diagnostic and prognostic performances of GLS1 was evaluated by the receiver operating characteristic curve and Cox regression model.ResultsThe sensitivity, specificity, positive predictive value, negative predictive value, Youden index, and area under the curve of GLS1 for the diagnosis of HCC were 0.746, 0.842, 0.979, 0.249, 0.588, and 0.814, respectively, which could be increased to 0.846, 0.886, 0.987,0.366, 0.732, and 0.921 when combined with glypican 3 (GPC3) and alpha-fetoprotein (AFP), indicating better diagnostic performance. Further, we developed a nomogram with GPC3 and GLS1 for identifying HCC which showed good discrimination and calibration. GLS1 expression was also related with age, T stage, TNM stage, Edmondson–Steiner grade, microvascular invasion, Ki67, VEGFR2, GPC3, and AFP expression in HCC. GLS1 expression was negatively correlated with disease-free survival (P < 0.001) probability of patients with HCC.ConclusionsIt was validated that GLS1 was a sensitive and specific biomarker for pathological diagnosis of HCC and had prognostic value, thus having practical value for clinical application.

Interaction between miR‑206 and lncRNA MALAT1 in regulating viability and invasion in hepatocellular carcinoma.

MicroRNAs (miRNAs) are strongly associated to the progression of hepatocellular carcinoma (HCC), which presents a high potential for diagnosis and treatment; however, the role of miRNAs is still largely unknown. The aim of the present study was to examine the expression and the biological role of miRNA (miR)-206 in the development of HCC, and to identify the underlying molecular mechanism. Results from this study show that miR-206 was significantly downregulated in HCC tissues and cell lines. It was observed that low expression of miR-206 was linked to advanced TNM stage, tumor nodularity and venous infiltration in patients with HCC; low miR-206 expression was associated with shorter survival times. miR-206 overexpression using miR-206 mimics notably decreased the proliferative ability and increased apoptosis of MHCC97-H and HCCLM3 HCC cell lines. Overexpression of miR-206 suppressed invasiveness associated with reduced epithelial-mesenchymal transition. Moreover, the c-Met oncogene, which is upregulated in HCC tissues, was negatively associated with the expression of miR-206. Notably, it was shown that miR-206 may exert its antitumor effect through suppressing c-Met/Akt/mTOR signaling. Low expression of miR-206 was shown to be regulated by lncRNA MALAT1 in HCC. Collectively, this study presented evidence that miR-206 was controlled by lncRNA MALAT1 and partially suppressed the proliferation and invasion of HCC through the c-Met/Akt/mTOR signaling pathway. According to these results, understanding MALAT1/miR-206-dependent regulation may lead to potential approaches for diagnosis and prospective treatment of HCC.

Polyphyllin I attenuates the invasion and metastasis via downregulating GRP78 in drug-resistant hepatocellular carcinoma cells.

Drug resistance to chemotherapy agents presents a major obstacle to the effective treatment of hepatocellular carcinoma (HCC), a common type of liver cancer. Increasing evidence indicates a link between drug resistance and the recurrence of HCC. Polyphyllin I (PPI), a promising pharmaceutical candidate, has shown potential therapeutic advantages in the treatment of sorafenib-resistant hepatocellular carcinoma (SR-HCC cells). In this study, we sought to investigate the mechanism underlying the inhibitory effect of PPI on the invasion and metastasis of SR-HCC cells. Our in vitro studies included scratch wound-healing migration assays and transwell assays to examine PPI's effect on HCC cell migration and invasion. Flow cytometry was employed to analyze the accumulation or efflux of chemotherapy drugs. The results of these experiments demonstrated that PPI increased the susceptibility of HCC to sorafenib while inhibiting SR-HCC cell growth, migration, and invasion. Molecular docking analysis revealed that PPI exhibited a higher binding affinity with GRP78. Western blot analysis and immunofluorescence experiments showed that PPI reduced the expression of GRP78, E-cadherin, N-cadherin, Vimentin, and ABCG2 in SR-HCC cells. Interference with and overproduction of GRP78 in vitro impacted the proliferation, migration, invasion, and metastasis of HCC cells. Further examination revealed that PPI hindered the expression of GRP78 protein, resulting in a suppressive effect on SR-HCC cell migration and invasion. Histological examination of tumor tissue substantiated that administering PPI via gavage to HepG2/S xenograft nude mice inhibited tumor growth and significantly reduced tumor size, as evidenced by xenograft experiments involving nude mice. Hematoxylin and eosin (HE) staining of tumor tissue specimens, along with immunohistochemistry (IHC), were conducted to evaluate the expression levels of Ki67, GRP78, N-cadherin, Vimentin, and ABCG2. The results indicated that PPI administration decreased the levels of proteins associated with metastasis and markers of drug resistance in tumor tissues, impeding tumor growth and spread. Overall, our findings demonstrated that PPI effectively suppressed the viability, proliferation, invasion, and metastasis of SR-HCC cells both in vitro and in vivo by modulating GRP78 activity. These findings provide new insights into the mechanism of PPI inhibition of SR-HCC cell invasion and metastasis, highlighting PPI as a potential treatment option for sorafenib-resistant HCC.

Effects of Ultrasound Contrast Agent-Encapsulated Paclitaxel Extract on the Proliferation and Invasion Ability of Hepatocellular Carcinoma Cells

Regarding to the limitations of paclitaxel (PTX) in cancer treatment, PTX was extracted from Taxus chinensis and PTX nano-ultrasound contrast agents (NUCA) were prepared to investigate their impacts on proliferation and invasion of hepatocellular carcinoma (HCC) cells (HCCCs). The PTX extract was obtained through extraction and multi-step purification methods using Taxus chinensis as the source material and poly(lactic-co-glycolic acid)-carboxylic acid (PLGA-COOH) as the experimental material. A modified double emulsion solvent evaporation (DESE) method was employed to prepare paclitaxel-loaded PLGA NUCA (PLGA@PTX). The particle size distribution (PSD) and zeta potential (ZP) of PLGA@PTX were identified using a laser particle size (PS) analyzer, while the drug-loading capacity (DLC) and encapsulation efficiency (EE) of PTX in PLGA@PTX NUCA were evaluated using high-performance liquid chromatography (HPLC). The in vitro release rate (IVRR) of PTX from PLGA@PTX NUCA was also analyzed. HepG2 lines, a human HCC cell line, were grouped into four randomly: a blank control group (Blank), a PTX group, a blank nano-contrast agent group without PTX encapsulation (PLGA), and a PTX-loaded NUCA group (PLGA@PTX). In the Blank group, HepG2 lines were cultured conventionally for 12 hours, while PTX or PLGA@PTX was added to the PTX and PLGA@PTX groups, respectively, to achieve a required concentration (10−7 mol/L) of PTX. An equal amount of PLGA nanoparticles was added to the PLGA group. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Transwell were utilized to judge the impacts of PLGA@PTX nanoparticles on proliferation and invasion of HepG2 lines, respectively. Moreover, flow cytometry (FCT) was utilized to examine the influence of PLGA@PTX nanoparticles on cell cycle (CC) and apoptosis of HepG2 lines. The results revealed that the purity of the PTX extract was as high as 99.04±0.92%. The average PS of PLGA@PTX NUCA was (432.79±4.56) nm, with a surface potential of (−10.79±2.28) mV. Furthermore, the EE and DLC were (89.27±2.63) % and (9.03±0.29) %, respectively. The inhibition rate (IR) to HepG2 lines and cell invasion and the apoptotic rate (AR) in the PLGA@PTX group were much higher to those in the PLGA and PTX groups (P &lt;0.01, P &lt;0.05). The ratio of G1/G0 phase in the CC was greatly lower in the PLGA@PTX group to the PLGA and PTX groups, showing obvious differences with (P &lt;0.05), while that of G2/M phase was higher (P &lt;0.05). These findings indicated that the prepared PLGA@PTX NUCA hindered the proliferation and invasion of HepG2 lines and induced CC arrest at the G2/M phase and apoptosis.

Tumor Microenvironment Composition and Related Therapy in Hepatocellular Carcinoma.

Globally, primary liver cancer is the third leading cause of cancer death, and hepatocellular carcinoma (HCC) accounts for 75%-95%. The tumor microenvironment (TME), composed of the extracellular matrix, helper cells, immune cells, cytokines, chemokines, and growth factors, promotes the immune escape, invasion, and metastasis of HCC. Tumor metastasis and postoperative recurrence are the main threats to the long-term prognosis of HCC. TME-related therapies are increasingly recognized as effective treatments. Molecular-targeted therapy, immunotherapy, and their combined therapy are the main approaches. Immunotherapy, represented by immune checkpoint inhibitors (ICIs), and targeted therapy, highlighted by tyrosine kinase inhibitors (TKIs), have greatly improved the prognosis of HCC. This review focuses on the TME compositions and emerging therapeutic approaches to TME in HCC.

AHSA1 Regulates Hepatocellular Carcinoma Progression via the TGF-β/Akt-Cyclin D1/CDK6 Pathway.

Activator of heat shock protein 90 (HSP90) ATPase Activity 1 (AHSA1) regulates proliferation, apoptosis, migration, and invasion of osteosarcoma and hepatocellular carcinoma (HCC). However, the novel mechanism of AHSA1 in the tumor biology of hepatocellular carcinoma (HCC) remains unclear. We analyzed AHSA1 expression in 85 pairs of clinical samples of HCC and the Cancer Genome Atlas database. The role of AHSA1 in HCC was proved by cell proliferation, colony formation, migration, cell cycle analysis in vitro, xenograft models and tumor metastasis assay in vivo, and bioinformatics. High AHSA1 expression was demonstrated in HCC and associated with invasive depth, clinical stage, and poor overall survival of patients. UnivariateCox analysis confirmed that AHSA1 was an independent prognostic factor for patients with HCC. Meanwhile, AHSA1 upregulation promoted cell proliferation, colony formation, and cell migration in vitro and tumor cell proliferation and metastasis of HCC cells in vivo. AHSA1 upregulation increased the cell cycle transition from G1 to S phase by increasing the expression of cyclinD1, cyclinD3, and cyclin-dependent kinase 6(CD). Transforming growth factor beta 1 (TGF-β1)-induced protein kinase B (Akt) signaling regulated the expression of downstream targets, including cyclinD1. AHSA1 expression was closely correlated with the expression of TGF-β, Akt, cyclinD1, cyclinD3, and CDK6 using the Gene Expression Profiling Interactive Analysis database. AHSA1 upregulation participated in HCC progression by regulating TGF-β/Akt-cyclinD1/CDK6 signaling. AHSA1 might serve as a biomarker for predicting the clinical outcome of patients with HCC. It is vital in tumor metastasis and disease progression of HCC and may facilitate the development of clinical intervention strategies against HCC.

Hepatocellular Carcinoma's Characteristics in an Endemic Country: A Closer Examination of Tumor Grade and Microvascular Invasion

Objective: Although histological grade and microvascular invasion are known predictors for patient survival and recurrence in hepatocellular carcinoma (HCC), their relationship with various clinical and histomorphological features of HCC remains unclear. Materials and Methods: Medical records were retrieved from 61 patients who were diagnosed with HCC from 2008-2018. Clinical and histomorphological variables that were hypothesized to be associated with histological grade and microvascular invasion were analyzed statistically using the Chi-square test or the Fisher’s exact test as alternatives. Multivariate analysis was performed with logistic regression model. Results: The majority of the patients had well to moderately-differentiated HCC (67.2%) with some of them presenting microvascular invasion (57.4%). Alpha-fetoprotein level (AFP) ≥100 ng/ml (p=0.036), tumor size &gt;7cm (p=0.031) and mitotic index ≥5 per 10 high power field (p=0.009) were significantly correlated with poorly-differentiated HCC. Mitotic Index ≥5 per 10 high power field was an independent factor for poorly differentiated HCC. Meanwhile BCLC stage B and mitotic index were also an independent factor for the presence of microvascular invasion. Conclusion: Larger tumor size and higher mitotic index was significantly correlated and independent factors for poorly differentiated HCC and microvascular invasion. In biopsy specimens for which the microvascular invasion is difficult to assess, histological grade, tumor size and mitotic index may be beneficial to depict the prognosis of patients with HCC.

LncRNA TCTN2 Promotes the Malignant Development of Hepatocellular Carcinoma via Regulating mIR-1285-3p/ARF6 Axis.

Hepatocellular carcinoma (HCC) is one of the most life-threatening malignant diseases. TCTN2 protein participates in tumorigenesis and development. However, whether lncRNA TCTN2 is associated with HCC pathogenesis remains unclear. The expression of lncRNA, TCTN2, miR-1285-3p, and ARF6 in HCC tissues and cells was detected by a quantitative Real-Time PCR (qRT-PCR) assay. lncRNA TCTN2-specific shRNA was transfected into HCC cells, and a functional investigation was performed. The direct interactions between lncRNA TCTN2 and miR-1285-3p and ARF6 were verified by dualluciferase reporter gene assay. A rescue experiment was performed to confirm the role of miR- 1285-3p/ARF6 in association with lncRNA TCTN2. LncRNA TCTN2 exhibited a high expression in HCC tumor tissues and cell lines. Knockdown of lncRNA TCTN2 suppressed cell proliferation and induced cell cycle arrest and apoptosis through regulating Cyclin D1/p21 and Bax/Bcl-2 signals. Meanwhile, the knockdown of lncRNA TCTN2 inhibited HCC cell migration and invasion through upregulating MMP2/MMP9. Mechanistic investigation revealed that lncRNA TCTN2 upregulated the expression of ARF6 via sponging miR-1285-3p. Rescue experiments indicated that miR-1285-3p inhibitor reversed the antitumor effects of lncRNA TCTN2 and ARF6 knockdown inhibited the progression of HCC. Our results suggested that the knockdown of lncRNA TCTN2 inhibited HCC development by regulating the miR-1285-3p/ARF6 axis, implying that the lncRNA TCTN2 is upregulated in HCC and may serve as a diagnostic biomarker in HCC. Furthermore, it may demonstrate an important value for the clinical treatment of patients with HCC.

Decreased B4GALT1 promotes hepatocellular carcinoma cell invasiveness by regulating the laminin-integrin pathway

Beta1,4-galactosyltransferases (B4GALTs) play a crucial role in several diseases, including cancer. B4GALT1 is highly expressed in the liver, and patients with mutations in B4GALT1 exhibit hepatopathy. However, the role of B4GALT1 in liver cancer remains unclear. Here, we found that B4GALT1 was significantly downregulated in hepatocellular carcinoma (HCC) tissue compared with the adjacent liver tissue, and low B4GALT1 expression was associated with vascular invasion and poor overall survival in patients with HCC. Additionally, silencing or loss of B4GALT1 enhanced HCC cell migration and invasion in vitro and promoted lung metastasis of HCC in NOD/SCID mice. Moreover, B4GALT1 knockdown or knockout increased cell adhesion to laminin, whereas B4GALT1 overexpression decreased the adhesion. Through a mass spectrometry-based approach and Griffonia simplicifolia lectin II (GSL-II) pull-down assays, we identified integrins α6 and β1 as the main protein substrates of B4GALT1 and their N-glycans were modified by B4GALT1. Further, the increased cell migration and invasion induced by B4GALT1 knockdown or knockout were significantly reversed using a blocking antibody against integrin α6 or integrin β1. These results suggest that B4GALT1 downregulation alters N-glycosylation and enhances the laminin-binding activity of integrin α6 and integrin β1 to promote invasiveness of HCC cells. Our findings provide novel insights into the role of B4GALT1 in HCC metastasis and highlight targeting the laminin-integrin axis as a potential therapeutic strategy for HCC with low B4GALT1 expression.

CircPSD3 is a promising inhibitor of uPA system to inhibit vascular invasion and metastasis in hepatocellular carcinoma

BackgroundVascular invasion is a major route for intrahepatic and distant metastasis in hepatocellular carcinoma (HCC) and is a strong negative prognostic factor. Circular RNAs (circRNAs) play important roles in tumorigenesis and metastasis. However, the regulatory functions and underlying mechanisms of circRNAs in the development of vascular invasion in HCC are largely unknown.MethodsHigh throughput sequencing was used to screen dysregulated circRNAs in portal vein tumor thrombosis (PVTT) tissues. The biological functions of candidate circRNAs in the migration, vascular invasion, and metastasis of HCC cells were examined in vitro and in vivo. To explore the underlying mechanisms, RNA sequencing, MS2-tagged RNA affinity purification, mass spectrometry, and RNA immunoprecipitation assays were performed.ResultscircRNA sequencing followed by quantitative real-time PCR (qRT-PCR) revealed that circRNA pleckstrin and Sect. 7 domain containing 3 (circPSD3) was significantly downregulated in PVTT tissues. Decreased circPSD3 expression in HCC tissues was associated with unfavourable characteristics and predicted poor prognosis in HCC. TAR DNA-binding protein 43 (TDP43) inhibited the biogenesis of circPSD3 by interacting with the downstream intron of pre-PSD3. circPSD3 inhibited the intrahepatic vascular invasion and metastasis of HCC cells in vitro and in vivo. Serpin family B member 2 (SERPINB2), an endogenous bona fide inhibitor of the urokinase-type plasminogen activator (uPA) system, is the downstream target of circPSD3. Mechanistically, circPSD3 interacts with histone deacetylase 1 (HDAC1) to sequester it in the cytoplasm, attenuating the inhibitory effect of HDAC1 on the transcription of SERPINB2. In vitro and in vivo studies demonstrated that circPSD3 is a promising inhibitor of the uPA system.ConclusionscircPSD3 is an essential regulator of vascular invasion and metastasis in HCC and may serve as a prognostic biomarker and therapeutic target.

Tripartite motif protein 6 promotes hepatocellular carcinoma progression via multiple pathways.

Hepatocellular carcinoma (HCC) ranks among the most prevalent malignancies worldwide and the third leading cause of cancer-related death. The TRIM (tripartite motif-containing) protein family members had been reported to be involved in carcinogenesis and tumor progression. Here we aimed to explore the expression profile of TRIM6 in HCC and investigate its clinical significance as well as underlying mechanisms. We retrospectively enrolled 138 HCC patients that underwent surgical resection in our hospital and tested protein expression level of TRIM6 through immunohistochemical staining. The correlation between TRIM6 and patients' characteristics was assessed by Chi-square test. Log-rank test and Cox hazard regression test were conducted for univariate and multivariate survival analyses, respectively. Two human HCC cell lines, Huh7 and Hep3B, were subjected for knockdown and overexpression assays, followed by phonotype tests including proliferation and invasion. Nude mice were used to generate xenograft model to validate our findings in vivo. TRIM6 was highly expressed in HCC specimen compared to nontumorous liver tissues. Higher TRIM6 expression was correlated with larger tumor size, later tumor stage, and worse prognosis. According to the cellular experiments, TRIM6-knockdown resulted in decreased expression of cyclin B1, c-Myc, Snail, MMP2, and VEGF-A. Consistently, TRIM6-knockdown led to impaired HCC proliferation, invasion, and angiogenesis. In contrast, TRIM6 overexpression showed opposite effects. Finally, the oncogenic role of TRIM6 in HCC was validated by in vivo mice experiments. TRIM6 can serve as a novel prognostic factor for HCC, which functions by multiple signaling pathways.