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1.
Aging (Albany NY) ; 11(23): 11565-11575, 2019 Dec 13.
Article in English | MEDLINE | ID: mdl-31831714

ABSTRACT

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.

2.
Biochem Biophys Res Commun ; 503(3): 1342-1348, 2018 09 10.
Article in English | MEDLINE | ID: mdl-30029875

ABSTRACT

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.


Subject(s)
Lung Neoplasms/metabolism , Lung Neoplasms/pathology , RNA, Long Noncoding/metabolism , Cell Proliferation , Female , Humans , Middle Aged , Proto-Oncogene Proteins c-myc/metabolism , Tumor Cells, Cultured
3.
Oncotarget ; 8(33): 54993-55002, 2017 Aug 15.
Article in English | MEDLINE | ID: mdl-28903397

ABSTRACT

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.

4.
Oncotarget ; 8(33): 55084-55093, 2017 Aug 15.
Article in English | MEDLINE | ID: mdl-28903404

ABSTRACT

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.

5.
Mol Med Rep ; 10(4): 1725-33, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25109274

ABSTRACT

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.


Subject(s)
Hepatic Stellate Cells/metabolism , Transcriptome , Animals , Carcinogenicity Tests , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line , Cell Movement , Cell Proliferation , Coculture Techniques , Female , Hepatic Stellate Cells/cytology , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Oligonucleotide Array Sequence Analysis , Rats , Rats, Inbred BUF , Transplantation, Homologous
6.
Oncol Rep ; 30(3): 1175-84, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23807027

ABSTRACT

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.


Subject(s)
Apoptosis , Carcinoma, Hepatocellular/pathology , Hepatic Stellate Cells/immunology , Liver Neoplasms/pathology , Lung Neoplasms/secondary , T-Lymphocytes/immunology , Animals , Blotting, Western , Carcinoma, Hepatocellular/immunology , Cell Proliferation , Dendritic Cells/immunology , Dendritic Cells/pathology , Flow Cytometry , Fluorescent Antibody Technique , Hepatic Stellate Cells/pathology , Immunoenzyme Techniques , Liver Neoplasms/immunology , Lung Neoplasms/immunology , Lymphocyte Activation , Rats , Rats, Inbred BUF , Rats, Inbred F344 , T-Lymphocytes/pathology , Tumor Cells, Cultured
7.
Mol Med Rep ; 5(3): 800-4, 2012 03.
Article in English | MEDLINE | ID: mdl-22160187

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement , Down-Regulation , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , MicroRNAs/antagonists & inhibitors , Neoplasm Invasiveness/pathology , Oligodeoxyribonucleotides, Antisense/metabolism
8.
Oncol Rep ; 25(3): 803-8, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21174062

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Matrix Metalloproteinase 2/physiology , Osteopontin/genetics , Urokinase-Type Plasminogen Activator/physiology , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Cell Line, Tumor , Down-Regulation/drug effects , Down-Regulation/physiology , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Genetic Therapy , Humans , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Male , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Metastasis , Oligodeoxyribonucleotides, Antisense/pharmacology , Oligodeoxyribonucleotides, Antisense/therapeutic use , Osteopontin/antagonists & inhibitors , Signal Transduction , Urokinase-Type Plasminogen Activator/metabolism , Xenograft Model Antitumor Assays
9.
Mol Biol Rep ; 38(6): 3671-7, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21104439

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Matrix Metalloproteinase 2/genetics , Osteopontin/metabolism , Up-Regulation/genetics , Urokinase-Type Plasminogen Activator/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Clone Cells , Collagen/metabolism , Drug Combinations , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Laminin/metabolism , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Matrix Metalloproteinase 2/metabolism , Neoplasm Invasiveness , Osteopontin/genetics , Plasmids/genetics , Proteoglycans/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transfection , Urokinase-Type Plasminogen Activator/metabolism
10.
J Exp Clin Cancer Res ; 29: 172, 2010 Dec 30.
Article in English | MEDLINE | ID: mdl-21190594

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Neoplasm Invasiveness/genetics , Osteopontin/biosynthesis , Proto-Oncogene Proteins c-myb/metabolism , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Electrophoretic Mobility Shift Assay , Gene Expression , Gene Expression Regulation, Neoplastic/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Neoplasm Invasiveness/pathology , Oligonucleotide Array Sequence Analysis , Osteopontin/genetics , Proto-Oncogene Proteins c-myb/genetics
11.
J Gastroenterol Hepatol ; 25(8): 1435-42, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20659235

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/chemistry , Carcinoma, Hepatocellular/surgery , Hepatectomy , Liver Neoplasms/chemistry , Liver Neoplasms/surgery , Osteopontin/analysis , Adult , Aged , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/secondary , China , Disease-Free Survival , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Recurrence, Local , Neoplasm Staging , Proportional Hazards Models , Risk Assessment , Risk Factors , Time Factors , Tissue Array Analysis , Treatment Outcome
12.
Zhonghua Zhong Liu Za Zhi ; 31(3): 164-9, 2009 Mar.
Article in Chinese | MEDLINE | ID: mdl-19615251

ABSTRACT

OBJECTIVE: Hepatic stellate cells (HSC) in hepatocellular carcinoma (HCC) transdifferentiate into extracellular matrix-producing myofibroblasts. Activated HSC can promote invasion and metastasis of HCC. To understand the differences of HSC in normal liver and HCC, we compared the gene expression patterns in HCC cell induction-activated and culture-activated rat HSC. METHODS: HSC were isolated by density centrifugation and exposed to conditioned medium from rat HCC cell line C5F. Expression of 22 012 genes in quiescent HSC, culture-activated HSC and HCC induction-activated HSC was analyzed by cDNA microarray and confirmed by real-time RT-PCR and Western blot. RESULTS: 1672 genes were differentially expressed in culture-activated HSC, including proinflammatory factors, cell adhesion molecules, cell surface receptors, signaling transduction molecules and immune factors. 711 genes were differentially expressed in HCC induction-activated HSC. Some of them were identical to those in culture-activated HSC. HCC Induction-activated HSC showed specific gene expression patterns, including Raf1, Rac2, Adam17, Wnt6, MMP-9 and TNF, suggesting that HCC cells can specifically induce HSC activation. CONCLUSION: The gene expression patterns in HCC induction-activated HSC are different from those in culture-activated HSC. HCC induction-activated HSC may play a major role in the invasion and metastasis of HCC. In vivo activation should be considered as the standard for the study of HSC biology. HCC induction-activated HSC should be considered as the standard for HSC biology studies.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Gene Expression Profiling , Hepatic Stellate Cells/metabolism , Liver Neoplasms/metabolism , Animals , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cells, Cultured , Gene Expression Regulation, Neoplastic , Hepatic Stellate Cells/pathology , Liver Neoplasms/pathology , Male , Oligonucleotide Array Sequence Analysis , Rats , Rats, Inbred F344
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