Causal effect of porphyria biomarkers on alcohol-related hepatocellular carcinoma through Mendelian Randomization.

PURPOSE: According to some cohort studies, an association exists between acute intermittent porphyria (AIP) and liver cancer. However, establishing a definitive causal relationship between porphyria and hepatocellular carcinoma (HCC) remains challenging. Prexisting studies regarding porphyria biomarkers and alcohol-related hepatocellular carcinoma (AR-HCC) make possible an entry point. In this study, we aimed to investigate the causal relationships between biomarkers of two types of porphyria, AIP and congenital erythropoietic porphyria (CEP), and AR-HCC. METHODS: Single-nucleotide polymorphisms (SNPs) associated with porphobilinogen deaminase (PBGD) and uroporphyrinogen-III synthase (UROS), along with outcome data on AR-HCC, were extracted from public genome-wide association studies (GWAS). The GWAS data were then used to explore the potential causal relationships via a two-sample Mendelian randomization (MR) analysis. The effect estimates were calculated using the random-effect inverse-variance-weighted (IVW) method. Additionally, the Cochrane's Q test, MR-Egger test, and leave-one-out analysis were conducted to detect heterogeneity and pleiotropy in the MR results. RESULTS: Using the IVW method as the primary causal effects model in the MR analyses, we found that both PBGD (effect estimate = 1.51; 95% CI, from 1.08 to 2.11, p = 0.016) and UROS (effect estimate = 1.53; 95% CI, from 1.08 to 2.18, p = 0.018) have a significant causal effect on AR-HCC. CONCLUSION: Our findings revealed a causal effect of both PBGD and UROS on AR-HCC, suggesting that both AIP and CEP have a causal association with AR-HCC.

PRDM16 from hepatic stellate cells-derived extracellular vesicles promotes hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) represents a lethal cancer, and most HCC cases occur in the fibrotic or cirrhotic livers. Hepatic stellate cells (HSCs), the main effector cells of liver fibrosis, could secret biological contents to maintain liver inflammation. Herein, we aimed to identify the key transcription factor secreted by extracellular vesicles (EVs) derived from HSCs and explored its oncogenic mechanism. The activated HSC cell line LX-2 was co-cultured with HCC cells with or without the EVs release inhibitor GW4869. The effects of co-culture with HSC on HCC cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition were analyzed. Co-culture with activated LX-2 enhanced HCC cell growth and motility, while GW4869 inhibited the pro-carcinogenic effect of HSC, suggesting that HSC promoted HCC progression through the secretion of EVs. HSC-derived EVs carried the key oncogenic transcription factor PRDM16, and uptake of EVs-derived PRDM16 by HCC cells activated the NOTCH1-mediated Notch signaling pathway. Knocking down PRDM16 in EVs or blocking Notch signaling in HCC cells significantly inhibited the tumor-promoting effects of HSC-derived EVs. Our study demonstrates that HSC-derived EVs activate the NOTCH1-mediated Notch signaling pathway in HCC cells by carrying PRDM16, leading to HCC progression.

COL15A1 interacts with P4HB to regulate the growth and malignancy of HepG2.2.15 cells.

Liver cancer is the fourth most common cause of cancer related deaths, ranking sixth in terms of incidence rate, and hepatocellular carcinoma (HCC) is the main type of liver cancer. Hepatitis B virus (HBV) infection is the main cause of HCC, and currently, HBV related HCC has become an important public health issue. COL15A1 encodes the alpha chain of collagen XV, a member of the FACIT collagen family, which has anti-angiogenic and anti-tumoral properties and play a vital role in tissue homeostasis in the liver, and its specific function in HBV-related HCC still needs further exploration. This study aimed to determine the regulatory role of COL15A1 in HBV-related HCC and explored the underlying mechanisms at the cellular level. Firstly, the biochip analysis results showed that the expression of COL15A1 was increased in human HBV-related HCC tissues. Furthermore, HBV induction also could significantly increase the expression of COL15A1 in hepatoma cell lines. Functionally, it found that COL15A1 silencing could significantly inhibit apoptosis and promote proliferation, migration, invasion and growth of HepG2.2.15. Mechanically, it found that COL15A1 could interact with P4HB，and its silencing could significantly increase the expression level of P4HB, thereby inhibiting the GRP76 expression and promoting growth and malignancy of HepG2.2.15 cells, revealing COL15A1 might play an anticancer role in HBV-related HCC.

Effects of forage type on the rumen microbiota, growth performance, carcass traits, and meat quality in fattening goats.

Forages fed to goats influence ruminal microbiota, and further contribute to affect growth performance, meat quality and its nutritional composition. Our objective for current study was to investigate the effects of different forages on growth performance, carcass traits, meat nutritional composition, rumen microflora, and the relationships between key bacteria and amino acids and fatty acids in the longissimus dorsi and semimembranosus muscles of goats. Boer crossbred goats were separately fed commercial concentrate diet supplemented with Hemarthria altissima (HA), Pennisetum sinese (PS), or forage maize (FG), and then slaughtered 90 days after the beginning of the experiment. Growth performances did not vary but carcass traits of dressing percentage, semi-eviscerated slaughter percentage, and eviscerated slaughter percentage displayed significant difference with the treatment studied. Meats from goats fed forage maize, especially semimembranosus muscles are rich in essential amino acids, as well as an increase in the amount of beneficial fatty acids. Our 16S rRNA gene sequencing results showed that the Firmicutes, Bacteroidetes, and Proteobacteria were the most dominant phyla in all groups but different in relative abundance. Further, the taxonomic analysis and linear discriminant analysis effect size (LEfSe) identified the specific taxa that were differentially represented among three forage treatments. The spearman's correlation analysis showed that rumen microbiota was significantly associated with the goat meat nutritional composition, and more significant positive correlations were identified in semimembranosus muscles when compared with longissimus dorsi muscles. More specifically, the lipid metabolism-related bacteria Rikenellaceae_RC9_gut_group showed positively correlated with meat amino acid profile, while genera Oscillospiraceae_UCG-005 were positively correlated with fatty acid composition. These bacteria genera might have the potential to improve nutritional value and meat quality. Collectively, our results showed that different forages alter the carcass traits, meat nutritional composition, and rumen microflora in fattening goats, and forage maize induced an improvement in its nutritional value.

Prognostic value of SPARC in hepatocellular carcinoma: A systematic review and meta-analysis.

OBJECTIVE: Hepatocellular carcinoma (HCC) is characterized by a high degree of malignancy, rapid proliferation of tumor cells, and early liver metastasis. Resistance to multiple drugs independent of the high expression of secreted protein acidic and rich in cysteine (SPARC) is associated with a high risk of recurrence and mortality. However, the prognostic value of SPARC in patients with HCC remains unclear. Therefore, we performed a meta-analysis to evaluate the relationship between the expression of SPARC and the prognosis of patients with HCC. METHODS: We searched for relevant articles in the CNKI, PubMed, EMBASE, and Web of Science databases. The 95% confidence intervals (CIs) were calculated for combined overall survival (OS) and disease-free survival (DFS) to assess the prognostic value of expression of SPARC in patients with HCC. RESULTS: In six of the studies, SPARC expression status was significantly associated with OS (combined hazard ratio [HR], 1.38; 95% CI, 1.0-1.82; Z = 2.27, P = 0.02) but not with DFS (combined HR, 0.79; 95% CI, 0.16-4.00, Z = 0.28, P = 0.78). Therefore, it cannot be assumed that upregulated SPARC expression has an effect on DFS in patients with HCC. CONCLUSION: Elevated SPARC expression is associated with a low survival rate but not with DFS in patients with HCC. Further studies are needed to confirm our conclusions. REGISTRATION: INPLASY registration number: INPLASY202180115. https://inplasy.com/inplasy-2021-8-0115/.

MIRLET7BHG promotes hepatocellular carcinoma progression by activating hepatic stellate cells through exosomal SMO to trigger Hedgehog pathway.

Hepatocellular carcinoma (HCC), commonly caused by liver fibrosis, is a global challenge with high morbidity. Activation of hepatic stellate cells (HSCs) contributes to hepatic fibrosis. Exosomes are small vesicles that play a significant role in cell-to-cell communication. Smoothened (SMO) is the key signal transducer for Hedgehog pathway. This study was designed to study the function and underlying mechanism of SMO in HSC activation. Functional assays including 5-Ethynyl-2´-deoxyuridine, colony formation, wound healing, transwell, and sphere formation assays disclosed the function of SMO. Western blot analysis of exosome biomarkers, immunofluorescence staining assay, electron microscope, and flow cytometry revealed the existence of exosomes. Bioinformatics analyses and mechanistic assays uncovered the interplays between RNAs. Nude mice xenograft model was established to evaluate HCC tumor growth. We uncovered that SMO was an oncogene in HCC cells and was low-expressed in quiescent HSCs. Then, SMO was upregulated in HSCs cultured with HCC cells-conditioned medium. Next, it was revealed that HCC cells-derived exosomes activated HSCs by transmitting SMO to HSCs. Subsequently, we recognized that microRNA let-7b host gene (MIRLET7BHG) served as the competing endogenous RNA against miR-330-5p to upregulate SMO. In turn, SMO induced hedgehog pathway to promote GLI family zinc finger 1 (Gli1), leading to transcriptional activation of MIRLET7BHG in activated HSCs. In summary, this study demonstrated that Gli1-induced MIRLET7BHG facilitated HCC by activating HSCs through exosomal SMO to stimulate hedgehog pathway, providing a new road for HCC treatment.

Nrf2 activation mediates tumor-specific hepatic stellate cells-induced DIgR2 expression in dendritic cells.

Our previous studies discovered that tumor-specific hepatic stellate cells (tHSCs) induced dendritic cell-derived immunoglobulin receptor 2 (DIgR2) expression in bone marrow-derived dendritic cells (mDCs), inhibiting splenic T cell activation. The current study aims to explore the underlying mechanism of DIgR2 expression by focusing on Nrf2 (nuclear-factor-E2-related factor 2) signaling. We show that tHSCs co-culture induced significant Nrf2 signaling activation in mDCs. The latter was evidenced by Nrf2-Keap1 disassociation, Nrf2 protein stabilization, accumulation and nuclear translocation. Expression of Nrf2-dependent genes, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), were detected in tHSCs-co-cultured mDCs. Importantly tHSCs-induced DIgR2 expression was blocked by Nrf2 shRNA or knockout (KO, by CRISPR/Cas9 method). Conversely, forced activation of Nrf2, by Keap1 shRNA or the Nrf2 activators (3H-1,2-dithiole-3-thione and MIND4-17), induced significant DIgR2 expression. tHSCs stimulation induced reactive oxygen species (ROS) production in mDCs. Conversely, ROS scavengers inhibited tHSCs-induced ROS production, Nrf2 activation and DIgR2 expression in mDCs. Significantly, tHSCs inhibited production of multiple cytokines (CD80, CD86 and IL-12) in mDCs, reversed by Nrf2 depletion. Moreover, Nrf2 shRNA or KO attenuated splenic T cell inhibition by tHSCs-stimulated mDCs. Together, we conclude that Nrf2 activation mediates tHSCs-induced DIgR2 expression in mDCs.

Long non-coding RNA EPIC1 promotes human lung cancer cell growth.

Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) is a MYC-interacting LncRNA. In the present study, the expression and potential function of Lnc-EPIC1 in human lung cancer cells are tested. We show that Lnc-EPIC1 expression is significantly higher in established/primary human lung cancer cells than that in human lung epithelial cells. Lnc-EPIC1 is also elevated in human lung cancer tissues. Silencing of Lnc-EPIC1 by targeted siRNA significantly inhibited human lung cancer cell growth, survival and proliferation, whiling inducing G1-S cell cycle arrest and cell apoptosis. MYC targets, including Cyclin A1, CDC20 and CDC45, were downregulated by Lnc-EPIC1 siRNA. MYC knockout by CRISPR/Cas-9 gene-editing method mimicked actions by Lnc-EPIC1 siRNA in A549 cells. Conversely, forced overexpression of Lnc-EPIC1 by a lentiviral construct increased expression of MYC targets to promote A549 cell growth. Lnc-EPIC1 directly associated with MYC protein in the nuclei of A549 cells. Significantly MYC knockout abolished Lnc-EPIC1 silencing- or overexpression-induced actions in human lung cancer cells. Together, our results show that Lnc-EPIC1 promotes human lung cancer cell growth possibly by targeting MYC.

DlgR2 knockdown boosts dendritic cell activity and inhibits hepatocellular carcinoma tumor in-situ growth.

Tumor-specific hepatic stellate cells (tHSCs) positively participate in human hepatocellular carcinoma (HCC) tumorigenesis and progression. Our previous studies have shown that tHSCs co-culture with dendritic cells (DCs) induced DIgR2 (dendritic cell-derived immunoglobulin receptor 2) expression. The latter is a member of IgSF inhibitory receptor suppressing DCs-initiated antigen-specific T-cell responses. In the current study, we show that hepatic artery injection of DlgR2 siRNA significantly inhibited in-situ HCC xenograft growth in rat livers. Further, 5-FU-medied inhibition of in-situ HCC growth was dramatically sensitized with DlgR2 silence. DlgR2 siRNA injection indeed downregulated DlgR2 in ex-vivo cultured tumor-derived DCs (tDCs). More importantly, tDCs activity was boosted following DlgR2 siRNA. These cells presented with upregulated CD80, CD86 and MHC-II. Production of interleukin-12 and tumor necrosis factor-α was also increased in the DlgR2-silenced tDCs. We propose that DlgR2 knockdown likely boosts the activity of tumor-associated DCs, and inhibits growth of in-situ HCC xenografts.

Tumor-specific hepatic stellate cells (tHSCs) induces DIgR2 expression in dendritic cells to inhibit T cells.

Tumor-specific hepatic stellate cells (tHSCs) contributes to tumorigenesis and progression of hepatocellular carcinoma (HCC). The potential function of tHSCs on dendritic cells (DCs) was studied here. We discovered that tHSCs co-culture induced upregulation of DIgR2 (dendritic cell-derived immunoglobulin receptor 2) in bone marrow-derived DCs (mDCs). Activation of MEK-ERK is required for DIgR2 expression in mDCs. MEK-ERK inhibitors or shRNA-mediated silence of MEK1/2 in mDCs inhibited tHSCs-induced DIgR2 expression. Meanwhile, tHSCs stimulation decreased production of multiple cytokines (CD80, CD86 and IL-12) in mDCs. Such an effect was almost reversed by DIgR2 shRNA in mDCs. Further, tHSCs-stimulated mDCs induced T-cell hypo-responsiveness, leading to decreased cytotoxic T lymphocyte (CTL) activity and reduced IFN-γ production in splenic T cells. T cell proliferation inhibition and apoptosis were also noticed. These actions on T cells were again largely inhibited by DIgR2 shRNA in mDCs. Together, our results indicate that tHSCs directly induces DIgR2 expression in DCs to inhibit T cells.

Chemotherapy regimen based on sorafenib combined with 5-FU on HIF-1α and VEGF expression and survival in advanced gastric cancer patients.

The present study investigated the effect of combined sorafenib chemotherapy on hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression and survival time of patients with advanced gastric cancer. From January 2010 to December 2011, 92 patients diagnosed with advanced gastric cancer were selected and randomly divided into the treatment group and control group. The treatment group was treated with sorafenib chemotherapy combined with 5-fluorouracil (5-FU), and the control group received 5-FU. The treatment course was 3-4 cycles. During the same period, 46 healthy persons admitted to the Second People's Hospital of Huaian were selected as the controls. A volume of 3-4 ml peripheral blood from each patient and control was collected before and after treatment. The expression levels of HIF-1α and VEGF in peripheral blood were measured by ELISA. The survival time of patients with advanced gastric cancer was followed and analyzed. Compared with healthy controls, serum levels of HIF-1α and VEGF were significantly higher in patients with advanced gastric cancer (P<0.05). After chemotherapy combined with sorafenib, the peripheral blood levels of HIF-1α and VEGF decreased significantly in the treatment group (P<0.05). The 5-year survival rate of patients in the two groups was followed. Compared with the control group, the 1-year survival rate of the treatment group was significantly higher (P<0.05). In conclusion, chemotherapy combined with sorafenib can effectively reduce serum levels of HIF-1α and VEGF in patients with advanced gastric cancer, and improve their 1-year survival rate and prognosis. Therefore, it has significant clinical application value.

Rat hepatic stellate cells alter the gene expression profile and promote the growth, migration and invasion of hepatocellular carcinoma cells.

The aim of the present study was to examine the effects of activated hepatic stellate cells (HSCs) and their paracrine secretions, on hepatocellular cancer cell growth and gene expression in vitro and in vivo. Differentially expressed genes in McA-RH7777 hepatocellular carcinoma (HCC) cells following non-contact co-culture with activated stellate cells, were identified by a cDNA microarray. The effect of the co-injection of HCC cells and activated HSCs on tumor size in rats was also investigated. Non-contact co-culture altered the expression of 573 HCC genes by >2-fold of the control levels. Among the six selected genes, ELISA revealed increased protein levels of hepatic growth factor, matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9). Incubation of HCC cells with medium conditioned by activated HSCs significantly increased the proliferation rate (P<0.001), migration rate and the number of invasive HCC cells (P=0.001). Co-injection of HCC cells and activated HSCs into rats significantly increased the weight of the resulting HCC tumors (P<0.01). The paracrine activity of activated HSCs markedly altered the gene expression profile of HCC cells and affected their growth, migration and invasiveness. The results from the present study indicate that the interaction between the activated HSCs and HCC has an important role in the development of HCC.

T-cell apoptosis induced by intratumoral activated hepatic stellate cells is associated with lung metastasis in hepatocellular carcinoma.

Profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro has recently been described in hepatocellular carcinoma (HCC). In the present study, we investigated the immune inhibitory activity of HSCs in vivo in an orthotopic rat HCC model with lung metastasis. Rats (n=24) were randomly sacrificed on days 7, 14, 21 and 28 (n=4 each). Lung tissues were stained with hematoxylin and eosin. Liver sections were stained for immunofluorescence analysis. T-cell apoptosis was detected using double staining for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Staining revealed marked and continuous accumulation of α-smooth muscle actin with tumor progression after orthotopic tumor implantation in rat liver. T lymphocyte numbers gradually increased following tumor progression, and subset analysis revealed an increase in the distribution of liver CD8+ and CD4+ T cells. Double staining for CD3 and TUNEL demonstrated T-cell apoptosis. Apoptotic T cells were more frequent in the HCC livers compared to the normal livers, and were spatially associated with intratumoral activated HSCs (tHSCs), suggesting a direct interaction. T-cell apoptosis was more frequently induced in the co-cultures of activated splenic T cells(aT)/tHSCs compared to aT/quiescent (q) HSCs or qT/tHSCs. tHSCs were positively correlated with T-cell apoptosis, and the percentage of T-cells undergoing apoptosis was positively correlated with the number of lung metastasis nodules. T-cell apoptosis may be promoted via an interaction with tHSCs, suggesting that tHSCs regulate T cells and contribute to lung metastasis in HCC.

Suppression of microRNA-96 expression inhibits the invasion of hepatocellular carcinoma cells.

MicroRNA-96 (miR-96) expression is dysregulated in certain types of cancers. However, the role of miR-96 in hepatocellular carcinoma (HCC) invasion and metastasis remains elusive. miR-96 expression was investigated in a number of HCC cell lines by quantitative reverse transcription­polymerase chain reaction (RT-PCR). Cell invasive ability of metastatic HCCLM6 cells transfected with anti-miR-96 oligonucleotides or miR-96 mimic was determined by Matrigel invasion assay in vitro. In addition, metastasis-associated protein, osteopontin, was evaluated by Western blotting. miR-96 expression was significantly increased in highly metastatic HCCLM6 cells. Decreasing miR-96 expression with anti-miR-96 oligonucleotides led to reduced migration and invasion of HCCLM6 cells in vitro. In particular, down-regulation of miR-96 decreased osteopontin expression in HCCLM6 cells. Suppression of miR-96 expression inhibits the invasion of HCC cells, suggesting that miR-96 may be a therapeutic target for inhibiting HCC invasion and metastasis.

Inhibition of T-cell responses by intratumoral hepatic stellate cells contribute to migration and invasion of hepatocellular carcinoma.

The stroma of hepatocellular carcinoma (HCC) is markedly infiltrated with activated hepatic stellate cells (HSCs), and associated invasion and metastasis of HCC. However, little is known of the role of HSCs in immune responses in HCC. The Buffalo rat HCC model was established. Quiescent HSCs (qHSCs) and intratumoral HSCs (tHSCs) were isolated. Surface molecules of tHSC were detected by flow cytometry, and gene expression was analyzed by fluorescence quantitative RT-PCR. T cell proliferation was monitored by [(3)H]-thymidine ((3)H-TdR) incorporation into DNA, and cytotoxic activity was assessed by measuring the release of (51)Cr. The level of cytokine expression by T cells was measured by enzyme-linked immunosorbent assay. T cell apoptosis was detected by double-stained terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and anti-CD3 antibodies. The migration and invasion of HCC was observed by transwell experiments. tHSCs express low levels of major histocompatibility complex (MHC) class I, MHC class II, and costimulatory molecules, and produce varying levels of cytokines. Addition of the tHSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation. The tHSC-induced T-cell hyporesponsiveness was associated with enhanced T-cell apoptosis, and contributed to the migration and invasion of hepatoma cell. tHSCs was associated with markedly enhanced expression of B7-H1. Blockade of B7-H1/PD-1 ligation significantly reduced HSC immunomodulatory activity, and hepatoma cell migration and invasion. tHSCs can induce T cell apoptosis, suggesting an important role for B7-H1. The interactions between tHSCs and T cells may contribute to hepatic immune tolerance and invasion and migration of HCC.

Down-regulation of osteopontin inhibits metastasis of hepatocellular carcinoma cells via a mechanism involving MMP-2 and uPA.

Osteopontin (OPN) has an important role in hepatocellular carcinoma (HCC) progression and metastasis. This study was to investigate the therapeutic potential of inhibition of OPN expression. A 2'-O-methoxyethylribose-modified phosphorothioate antisense oligonucleotides (ASO) was used to knock-down OPN expression in the human metastatic HCC cell line HCCLM6 and in nude mice orthotopically implanted with HCCLM6 showing highly spontaneous lung metastasis. Furthermore, we assessed the metastatic potential of HCCLM6 cells in vitro and in vivo after ASO treatment. Treatment of HCCLM6 cells with OPN ASO inhibited OPN mRNA expression in a dose- and time-dependent manner, whereas the control oligonucleotides had no effect. OPN ASO significantly suppressed migration and invasion of HCCLM6 cells in vitro. Specific suppression of OPN also inhibited matrix metalloproteinase 2 (MMP-2) and urokinase-type plasminogen activator (uPA) expression in HCCLM6 cells. In mice bearing orthotopical xenografts with HCCLM6, OPN inhibition following therapeutic treatment with OPN ASO significantly decreased lung metastases although tumor weight did not appear to be reduced. These findings suggest that OPN-targeted therapy may be a promising strategy for the treatment of HCC metastases.

Osteopontin promotes hepatocellular carcinoma invasion by up-regulating MMP-2 and uPA expression.

Osteopontin (OPN) is over-expressed in a variety of cancers, but its role in hepatocellular carcinoma (HCC) progression has not been clarified. In this study, weakly tumorigenic, non-metastastic human HCC cell line SMMC-7721 cells were forced to over-express OPN via stable transfection. A series of functional assays were performed to assess the effects of OPN on tumor cell behaviors and cDNA microarray was used to identify the genes regulated by OPN. The results showed that OPN significantly enhanced the migration and invasion of SMMC-7721 cells in vitro. In addition, CD44v6 antibody could significantly inhibit the invasion of OPN over-expressing SMMC-7721 cells. Moreover, MMP-2 and uPA expressions were significantly up-regulated in OPN over-expressing SMMC-7721 cells. Together, these findings indicate that OPN enhanced HCC cells invasion through interaction with its receptor CD44v6 and increased MMP-2 and uPA expressions, providing at least one mechanism for OPN-mediated HCC progression and metastasis.

Transcription factor c-Myb promotes the invasion of hepatocellular carcinoma cells via increasing osteopontin expression.

BACKGROUND: Specific gene expression is tightly regulated by various transcription factors. Osteopontin (OPN) is a phosphoprotein that mediates hepatocellular carcinoma (HCC) progression and metastasis. However, the mechanism of OPN up-regulation in HCC metastasis remains to be clarified. METHODS: Oligonucleotide array-based transcription factor assays were applied to compare different activities of transcription factors in two human HCC cell lines with different OPN expression levels. The effects of one selected transcription factor on OPN expression were further evaluated. RESULTS: Eleven transcription factors were over-expressed in metastatic HCC cell line HCCLM6 cells whereas twelve transcription factors were down-regulated. Electrophoretic mobility shift assays (EMSA) and reporter gene assays showed that one of up-regulated transcription factors c-Myb could bind the OPN promoter and increase its transcription activity. In addition, small interfering RNA targeting c-Myb could inhibit OPN expression and significantly decrease migration and invasion of HCCLM6 cells in vitro. CONCLUSION: Our data first demonstrate that c-Myb has a functionally important role in the regulation of OPN expression in HCC cells, suggesting that c-Myb might be a new target to control HCC metastasis.

Osteopontin, a single marker for predicting the prognosis of patients with tumor-node-metastasis stage I hepatocellular carcinoma after surgical resection.

BACKGROUND AND AIM: Osteopontin (OPN) has been linked to clinical outcomes in several solid tumors. However, it has not been fully evaluated whether OPN could be used as a single marker for the prognosis of patients with hepatocellular carcinoma (HCC), particularly in patients of the tumor-node-metastasis (TNM) stage I. METHODS: A total of 151 patients with HCC who underwent surgical resection were enrolled, including 112 patients of the TNM stage I. OPN expression was evaluated using immunohistochemistry in the tissue microarrays derived from these patients. Immunoreactivity was classified according to the percentage and intensity of staining: negative (-), weak (+) and strong (++). The impact of OPN expression on survival of patients was analyzed. RESULTS: In total, 65.6% (99 of 151) of HCC tissues expressed OPN. Overall survival in patients of OPN (-) group was significantly higher than those of OPN (+) or OPN (++) group (P = 0.049 and P = 0.001). Interestingly, in patients of the TNM stage I, OPN expression was correlated with the early recurrence after surgical resection (P = 0.001). Multivariate analysis showed that OPN expression was an independent prognostic factor for overall survival and disease-free survival in patients with the TNM stage I HCC (hazard ratio, 2.272, P = 0.014 and 1.982, P = 0.037). CONCLUSIONS: These results suggest that OPN is commonly expressed in HCC and is a useful marker for predicting the prognosis of patients with the TNM stage I HCC, contributing to determining which individual patient needs adjuvant therapy to prevent the early recurrence after surgical resection.

Gene expression profiles during activation of cultured rat hepatic stellate cells by tumoral hepatocytes and fetal bovine serum.

PURPOSE: Hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. Myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma (HCC). In this study, we determined gene expression changes in two different models of HSC activation, induction-activated HSCs (iHSCs) and culture-activated HSCs (cHSCs). METHODS: Hepatic stellate cells were isolated by density centrifugation and exposed to conditioned medium (CM) from the rat HCC cell line C5F, and fetal bovine serum (FBS). Expression of 27,100 genes in quiescent HSCs, cHSCs and iHSCs was analyzed by microarray and was confirmed on a subset of genes by real-time RT-PCR and Western blot. RESULTS: One thousand nine hundred sixty-seven genes were differentially expressed in cHSCs and iHSCs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors such as Il1a, Vcam1, Ccl6, Ilr7, PRAP, osteopontin, Gp39, Raf1, Rac2, Adam17, Wnt6, MMP-9, and Cfd. C5F-CM-induced activation only partially reproduced the gene expression changes observed during FBS culture activation. iHSCs showed specific gene expression, suggesting that HCC cells can specifically induce HSC activation. CONCLUSIONS: Induction- activated HSCs' gene expression patterns were partially similar to and different from that of cHSCs. iHSCs might play an important role in invasion and metastasis of HCC. This study provided theoretical foundations for investigating the biology of HSCs in HCC.