Search

BackgroundTransarterial chemoembolization (TACE) stands for the most commonly utilized therapy for hepatocellular carcinoma (HCC) worldwide. This study was to explore the potential predictive and prognostic roles of LAG-3 and PD-L1 as serum biomarkers in HCC patients underwent TACE treatment.MethodsA total of 100 HCC patients receiving TACE as well as 30 healthy controls were enrolled in the study. Serum LAG-3 and PD-L1 levels were determined at baseline and 3 day after TACE using enzyme-linked immunosorbent assay (ELISA).ResultsWe found serum levels of LAG-3 and PD-L1 were significantly elevated in HCC patients compared with healthy controls. Interestingly, patients with low pre-TACE and post-TACE levels of LAG-3 but not PD-L1 had a high probability of achieving an objective response (OR) after TACE treatment. Additionally, high pre-TACE LAG-3 level was correlated with poor disease outcome, and the patients with both high serum LAG-3 and PD-L1 level had the shorter overall survival (OS) than patients who are either PD-L1 or LAG-3 high or both PD-L1 and LAG-3 low. High pre-TACE serum LAG-3 level was positively associated with more cirrhosis pattern, advanced BCLC stage, pre-TACE alanine aminotransferase (ALT) level, and pre-TACE aspartate aminotransferase (AST) level. Furthermore, in 50 patients who underwent TACE, the serum LAG-3 level was significantly decreased at 3 day after TACE.ConclusionBoth pre-TACE and post-TACE serum LAG-3 levels could serve as powerful predictors for tumor response of TACE, and high pre-TACE serum LAG-3 level was an indicator for poor prognosis in HCC.

Tumor mutation burden (TMB) is associated with immune infiltration, while its underlying mechanism in hepatocellular carcinoma (HCC) remains unclear. A long noncoding RNA (lncRNA)-related competitive endogenous RNA (ceRNA) network can regulate various tumor behaviors, and research about its correlation with TMB and immune infiltration is warranted. Data were downloaded from TCGA and ArrayExpress databases. Cox analysis and machine learning algorithms were employed to establish a lncRNA-based prognostic model for HCC. We then developed a nomogram model to predict overall survival and odds of death for HCC patients. The association of this prognostic model with TMB and immune infiltration was also analyzed. In addition, a ceRNA network was constructed by using DIANA-LncBasev2 and the starBase database and verified by luciferase reporter and colocalization analysis. Multiplex immunofluorescence was applied to determine the correlation between ULBP1 and PD-L1. An eight-lncRNA (SLC25A30-AS1, HPN-AS1, LINC00607, USP2-AS1, HCG20, LINC00638, MKLN1-AS and LINC00652) prognostic score model was constructed for HCC, which was highly associated with TMB and immune infiltration. Next, we constructed a ceRNA network, LINC00638/miR-4732-3p/ULBP1, that may be responsible for NK cell infiltration in HCC with high TMB. However, patients with high ULBP1 possessed a poorer prognosis. Using multiplex immunofluorescence, we found a significant correlation between ULBP1 and PD-L1 in HCC, and patients with high ULBP1 and PD-L1 had the worst prognosis. In brief, the eight-lncRNA model is a reliable tool to predict the prognosis of HCC patients. The LINC00638/miR-4732-3p/ULBP1 axis may regulate immune escape via PD-L1 in HCC with high TMB.

Background Fibrinogen-like protein 1 (FGL1)—Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. Methods The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8+T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied. Results We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3+cells but not PD-L1. CD8+ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3+cells and low levels of CD8+ T cells were correlated with poor disease outcome. Moreover, LAG-3+cells deteriorated patient stratification based on the abundance of CD8+ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC+) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC−). Furthermore, PD-L1 TC− in combination with high densities of LAG-3+cells showed the worst prognosis, and PD-L1 TC+ patients with low densities of LAG-3+cells had the best prognosis. Conclusions LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3+cells and CD8+ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

Three carbapenem-resistant clinical isolates of the Enterobacter cloacae complex (ECC) were recovered from different patients in a hospital. All 3 isolates carried 2 carbapenemase genes blaKPC-2 and blaNDM-1. A study was performed to characterize their relatedness and to investigate possible links among the patients. Whole genome sequencing revealed that the isolates were Enterobacter hormaechei and belonged to ST177 of the ECC. There were 19-142 single nucleotide polymorphisms (SNPs) between the isolates, suggesting that the isolates were likely from a central reservoir, which might have existed for some time. blaKPC-2 and blaNDM-1 were carried on 2 different IncF-type plasmids in the isolates. The 3 blaNDM-1-carrying plasmids were almost identical and were self-transmissible, while the blaKPC-2-carrying plasmids were only transmissible in the presence of the blaNDM-1-carrying plasmid. The source of and direct links among them were not identified, suggesting a hospital transmission of a common multidrug resistant strain.

Cytokines play an important role in the development of tumors. The purpose of the present study was to evaluate the mechanisms and cytokine level changes after transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC). The Short Time-series Expression Miner (STEM) program was utilized to cluster cytokine expression profiles from the day before TACE to day 21 post-TACE. Based on the identified significant signatures, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. Cytokines were serially monitored in 60 evaluable patients to identify the results of the STEM program. Examination of the significant signatures identified 6 significant time-varied expression patterns for 507 cytokines (profiles 16, 18, 28, 41, 42 and 43). GO analysis was enriched in ‘cytokine receptor-binding’ and ‘cytokine receptor activity’, and the identified signaling pathways included ‘cytokine-cytokine receptor interaction’ and the ‘JAK-STAT signaling pathway’. Ciliary neurotrophic factor (CNTF) level was increased early after TACE, reaching a peak on day 7 before finally decreasing from day 14 onwards, and was significantly positively correlated with aminotransferase level. Serum levels of pre-TACE IL-10 predicted the local tumor response and overall survival (OS) of the patients, while serum levels of post-TACE IL-1β only indicated the local tumor response of the patient. Overall, the present study identified cytokine time-series expression profiles of patients with HCC undergoing TACE. Early phase increases in CNTF after TACE were associated with post-treatment hepatic injury. IL-1β may reflect an objective response after TACE, while IL-10 may represent a biomarker for OS and the objective response pre-TACE, which may help patients with HCC to benefit from TACE.

CAPS1 (calcium-dependent activator protein for secretion) is a multi-domain protein involved in regulating exocytosis of synaptic vesicles and dense-core vesicles. However, the expression and function of CAPS1 in cholangiocarcinoma (CCA) remains unclear. In the present study, we explored the role of CAPS1 in CCA carcinogenesis. CAPS1 expression was explored using western blotting and immunohistochemistry in four CCA cell lines and clinical samples from 90 cases of CCA. The clinical significance of CAPS1 was analyzed. The biological function of CAPS1 in CCA cells was detected in vitro and in vivo. The underlying mechanism of CAPS1 function was explored by detecting the expression of critical molecules in its associated signaling pathways. The mechanism of CAPS1 downregulation in tumor tissues was explored using in silico prediction and luciferase reporter assays. CAPS1 expression was reduced in CCA cell lines and human tumor tissues. Loss of CAPS1 in tumor tissues was closely associated with poor prognosis of patients with CCA. Moreover, CAPS1 expression correlated significantly with tumor–node–metastasis stage, lymph node metastasis, and vascular invasion. Lentivirus-mediated CAPS1 overexpression substantially prevented clone formation, cell proliferation, and cell cycle progression. CAPS1 overexpression also suppressed carcinogenesis in nude mice. Mechanistically, CAPS1 overexpression greatly accelerated the ERK and p38 MAPK signal pathways. In addition, microRNA miR-30e-5p negatively regulated CAPS1 expression. These data showed that CAPS1 functions as a tumor suppressor in CCA. Reduced CAPS1 expression could indicate poor prognosis of patients with CCA.

Ethnopharmacological relevance Many studies have confirmed that traditional Chinese herbs exert potential anti-tumor effects. Actinidia Chinensis Planch root has been used as a traditional Chinese medicine (TCM) for thousands of years. However, the mechanism of anti-tumor effects of Actinidia Chinensis Planch root has not been clearly clarified. Aim of the study To explore the molecular biological mechanisms underlying the inhibitory effect of Actinidia Chinensis Planch root extract (acRoots) on hepatocellular carcinoma (HCC). Materials and methods In our previous study, we used mRNA chip analyses to identify genes regulated by acRoots. Further analyses of altered genes led to the identification of a key regulator of genes that responds to acRoots. We explored the effects of acRoots on the proliferation and invasion of HCC cells via cell counting as well as transwell assays, and further explored the molecular mechanisms underlying the effects of acRoots on HCC cells using qRT-PCR, western blot, and Chip-PCR. Results Increasing the concentration of acRoots as well as prolonging its action time enhanced the inhibitory activity of acRoots as well as its cytotoxicity against HCC cells. High TARBP2 expression in HCC cells, which is associated with advanced-stage HCC and poor prognoses in HCC patients, was downregulated by treatment with acRoots. Furthermore, acRoots inhibited proliferation, invasion, and epithelial-to-mesenchymal transition by downregulating TARBP2 expression. HCC cells with higher TARBP2 expression were more sensitive to acRoots. The expression of TARBP2 and DLX2 in HCC patients and HCC cell lines was significantly positively correlated, and DLX2 as a transcription factor may promote TARBP2 expression, thereby further activating the JNK/AKT signaling pathway leading to the inhibition of HCC. Conclusions acRoots inhibited the malignant behavior of HCC cells by inhibiting TARBP2 expression, which is affected by the transcription factor DLX2, leading to a reduction in JNK/AKT signaling pathway activation.

TIPE1 (tumor necrosis factor-α-induced protein 8-like 1) contributes to cell death in diverse cancers. However, the expression and biological functions of TIPE1 in colon cancer remain unclear. In the present study, we report that TIPE1 was downregulated in colon cancer tissues and positively correlates with prognosis of colon cancer patients. TIPE1 overexpression significantly inhibits colon cancer cell growth both in vitro and in vivo through impairing stemness, accompanied with downregulation of the stemness-related markers, ALDH, CD133, CD44 and SOX-9. Mechanically, TIPE1 directly targets β-catenin and promotes β-catenin degradation in a protease-dependent manner, and Wnt/β-catenin signaling plays a crucial role during TIPE1-mediated stemness inhibition in colon cancer. These findings reveal that TIPE1 exerts anti-tumor effects in colon cancer and suggest that TIPE1 would be a therapeutic target for cancers.

Objectives LncRNA nuclear‐enriched abundant transcript 1 (NEAT1) participates in the development and progression of multiple malignancies. However, the molecular mechanism by which NEAT1 contributes to colorectal cancer (CRC) remains unclear. Methods The association between lncRNA NEAT1 expression and clinicopathological characteristics and prognosis in patients with CRC was analysed by TCGA RNA‐sequencing data. MTT, colony formation, flow cytometry, transwell assays and a xenograft tumour model were used to assess the functions of NEAT1. Bioinformatics and spearman correlation analysis were used to identify the NEAT1‐specific binding with miRNAs, and luciferase gene report and RIP assays were performed to confirm the interaction between miR‐193a‐3p (miR‐193a) and NEAT1 in CRC cells. Results Upregulation of NEAT1 expression was significantly correlated with TNM stage, poor survival and tumour recurrence in patients with CRC, and acted as an independent prognostic factor for tumour recurrence. Knockdown of NEAT1 suppressed cell proliferation, colony formation abilities and invasive potential and induced cell apoptosis, but overexpression of NEAT1 reversed these effects. Furthermore, NEAT1 was confirmed to act as a sponge of miR‐193a, and knockdown of NEAT1 attenuated miR‐193a inhibitor‐induced tumour promoting effects and L17RD expression in CRC cells. miR‐193a harboured negative correlation with NEAT1 and IL17RD expression in CRC specimens. In vivo experiment further validated the inhibitory effects of NEAT1 knockdown on xenograft tumour growth. Conclusion Our findings demonstrate that lncRNA NEAT1 acts as an oncogenic role in CRC cells by sponging miR‐193a and may represent a potential marker for CRC patients.

Aim Phospholipase C-γ1 (PLCG1) was previously found to be involved in a variety of oncogenic behaviors such as cell motility, cell proliferation, cell migration, and invasion. However, its function in hepatocellular carcinoma (HCC) was unknown. Here, we explored the expression pattern and function of PLCG1 in HCC progression. Methods Expression of PLCG1 was examined by western blotting in hepatoma cells and human tumor tissues. Expression was also detected by immunohistochemistry in 150 HCC clinical samples, and its clinical significance was analyzed. The influence of PLCG1 on HCC carcinogenesis were determined in vitro and in vivo. The underlying mechanisms were explored by detecting the expression of critical molecules of signaling pathways. Results The results showed that PLCG1 was overexpressed in hepatoma cell lines and clinical HCC tissues. Increased PLCG1 expression in tumor tissues was remarkably correlated with poor clinical features of HCC. Patients with positive PLCG1 expression in tumor tissues had shorter overall survival and relapse-free survival. Phospholipase C gamma 1 could substantially promote cell proliferation, anchor growth, and cell invasion in vitro. The in vivo study showed that inhibition of PLCG1 in hepatoma cells significantly repressed tumor growth in nude mice. Furthermore, we showed that PLCG1 might exert its function by activating the mitogen-activated protein kinase and nuclear factor-κB signaling pathways. Conclusion Our data indicated that PLCG1 could act as an oncogene in HCC carcinogenesis and could serve as a valuable prognostic marker and potential therapeutic target for HCC.

Background Hepatocellular carcinoma (HCC) develops in a complex microenvironment characterized by chronic inflammation. In recent years, cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology. This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells. Methods Human HCC cells HepG2 and Huh7 were cultured and stimulated with lipopolysaccharide (LPS) for 24 h. The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations, cholesterol uptake, and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), LDL receptor (LDLR), sterol regulatory element-binding transcription factor 2 (SREBF2), and proprotein convertase subtilisin/kexin 9 (PCSK9) were comparatively analyzed. Simultaneously, the effects of nuclear factor-kappa B (NF-κB) signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha (IKKα) and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3 (TAB3) via RNAi and microRNA (miR)-195. Subsequently, the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated. Results Pro-inflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR, LDLR, and SREBF2, while downregulating the expression of PCSK9. These effects were revealed to depend on NF-κB signaling pathway by knocking-down and overexpression of IKKα and TAB3. Additionally, miR-195, a regulator directly targeting IKKα and TAB3, blocked the effects of cholesterol accumulation, further supporting the critical role of pro-inflammation NF-κB signaling in regulating cholesterol accumulation. Intriguingly, the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-κB signaling pathway. Conclusions These results indicated that pro-inflammation effects of NF-κB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.

The development of hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species. However, the importance and biologic functions of p66Shc in HCC are unclear. The clinical significance of p66Shc was assessed in a large cohort of patients with HCC. High Shc1 expression was closely correlated with poor clinical outcomes and early recurrence of HCC. p66Shc expression was also determined in HCC samples and cell lines and found to be increased. Moreover, knockdown of p66Shc significantly inhibited cell proliferation, motility in vitro and tumor growth in vivo and could attenuate the proliferation, and motility of cells stimulated by activated macrophage conditioned media. Mechanically, p66Shc knockdown inhibited phosphorylation of STAT3 on serine 727 in vitro and in vivo. Our results show that high p66Shc expression in HCC predicts a worse prognosis for survival. Furthermore, p66Shc may serve as a novel candidate target for HCC therapy.

bHLH/PAS proteins play important roles in tumor progression. Lost or reduced expression of single-minded homolog 2 (SIM) as well as aryl hydrocarbon receptor repressor (AHRR) has been observed in cancerous human tissues. Here, we investigated the role of aryl hydrocarbon receptor nuclear translocator (ARNT), another bHLH/PAS protein, in hepatocellular carcinoma (HCC). Using tissue microarray and immunohistochemistry, we found that intratumoral ARNT was inversely correlated with time to recurrence and overall survival of HCC patients after resection. Knockdown of ARNT in HepG2, HCCLM3 and HCCLM6 cells significantly shortened cell doubling time, increased S-phase cell populations and accelerated in vivo HCCLM6 growth and metastasis. After ARNT expression was rescued, prolonged cell doubling time and decreased S-phase cell populations were observed in HepG2, HCCLM3 and HCCLM6 cells. And, HCCLM6 growth and metastasis in vivo were remarkably inhibited. Screening by quantitative reverse-transcription PCR and PCR arrays revealed that cyclin E1, CDK2, Fos and Jun were negatively regulated by ARNT, whereas CDKN1C, CNKN2A, CDKN2B, MAPK11 and MAPK14 were positively regulated in HCC. According to the results of immunoprecipitation assay, both ARNT/ARNT and ARNT/AHRR complexes were clearly formed in HCCLM6 xenograft with increased ARNT expression. In summary, ARNT is an important regulator of HCC growth and metastasis and could be a promising prognostic candidate in HCC patients.

To study biological characteristics of stable red fluorescent protein (RFP)-expressing or green fluorescent protein (GFP)-expressing HCCLM3 cell lines and those of their relevant xenograft models in nude mice.HCCLM3, a human hepatocellular carcinoma cell line with high metastatic potential was infected with RFP or GFP full-length cDNA via lentivirus. Stable RFP-expressing or GFP-expressing HCCLM3 cells, namely HCCLM3-R and HCCLM3-G, were subcutaneously injected and two patient-like metastatic models of HCCLM3-R and HCCLM3-G in nude mice were established using surgical orthotopic implantation from subcutaneous tumor tissues. Cell proliferation, karyotype, biomarker expression, tumor growth, and metastasis of HCCLM3-R and HCCLM3-G were analyzed in vitro and in vivo.RFP and GFP genes were integrated in genomic DNA of HCCLM3. HCCLM3-R and HCCLM3-G expressed red and green fluorescence, stable and intense, 300 days after 60 consecutive passages, and also positively expressed CK8+, P16+, AFP+ and negatively expressed HBsAg-. Their biomarker expression and karyotype were found to be similar to those of the parental HCCLM3, and their tumorigenesis occurred in 10 nude mice without exception after a subcutaneous injection and did the same in 20 nude mice after an orthotopic implantation. The results showed that the rate of spontaneous metastasis to the liver and lung and peritoneal seeding was 100, 100, and 90%, respectively.Stable fluorescent protein-expressing HCCLM3-R and HCCLM3-G xenografts in nude mice could be of two useful models for studying mechanisms of hepatocellular carcinoma growth and metastasis in real time.

Inflammatory factors play a vital role in the progression of liver cancer, although exact factors and related mechanisms still remain unclear. The present study aimed at screening inflammatory factors related to liver cancer metastasis and investigating the potential mechanism by which cancer cells are recruited. We screened and validated inflammatory factors by microarray and RT-PCR. Small interfering RNA (siRNA) and recombinant protein were used to assess CXCL5 effects on the movement of liver cancer cells (LCCs). Our screening microarray demonstrated over-expression of CXCL5 in LCCs with high metastatic potentials. CXCL5 increased LCCs migration and invasion, probably through autocrine and paracrine mechanisms. CXCL5-CXCR2 and ERK1/2 pathways could play critical roles in the regulation of LCCs migration. Our data indicates that LCCs per se may act as the producer and receptor of CXCL5 responsible for liver cancer migration and invasion.