膜型基质金属蛋白酶-1与肝细胞癌侵袭转移性的关系

目的 探索膜型基质金属蛋白酶－１（ｍｅｍｂｒａｎｅ－ｔｙｐｅ１ｍａｔｒｉｘ ｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ,ＭＴ１－ＭＭＰ）与肝细胞癌（ＨＣＣ）侵袭转移性的关系及以ＭＴ１－ＭＭＰ来判断肝细胞癌侵袭转移性的可能性和方法。方法 通过ＲＴ－ＰＣＲ对正常肝、ＨＣＣ及其癌旁组织、癌栓组织,裸鼠人肝癌高、低转移模型中的ＭＴ１－ＭＭＰｍＲＮＡ进行半定量研究,并与ＨＣＣ侵龚转移的病理指标进行统计分析,结果 正     

circZFR promotes cell proliferation and migration by regulating miR-511/AKT1 axis in hepatocellular carcinoma

BackgroundEmerging data suggest the crucial regulatory roles of circular RNAs (circRNAs) in hepatocellular carcinoma (HCC). However, the pathophysiology role of circZFR in HCC remains largely unknown.AimsThis study aims to disclose the functions of circZFR in HCC progression and its potential molecular mechanism.MethodscircZFR and miR-511 were identified by qRT-PCR. Colony formation assay, wound-healing assay, transwell assay, and flow cytometry assay were performed to determine the cell proliferation, migration, invasion and apoptosis. Western blotting and immunohistochemistry (IHC) were utilized to evaluate the expression level of AKT1, GSK3β, β-catenin and cascades of proliferation-related proteins both in vitro and in vivo. Dual luciferase reporter assay was conducted to evaluate the interactions among circZFR, miR-511 and AKT1.ResultsThe expression of circZFR was enhanced and the expression of miR-511 was down-regulated in HCC tissues and cells. Functionally, circZFR silencing or miR-511 overexpression suppressed cell proliferation, migration and invasion, and induced apoptosis of HCC cells. Mechanistically, circZFR acted as a miR-511 sponge to up-regulate its target gene AKT1, which activated cascades of proliferation-related proteins (c-Myc, cyclin D1, Survivin and Bcl-2). Furthermore, depletion of circZFR inhibited tumorigenesis and decreased the expression level of AKT1 in xenograft models.ConclusioncircZFR promotes HCC progression by directly down-regulating miR-511 to activate AKT1 signaling, suggesting that circZFR is a potential target in HCC treatment. Targeting circZFR may provide therapeutic benefits for HCC.     

Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of beta1 integrins.

Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin beta1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin beta1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin beta1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of beta1 integrins.     

Peroxiredoxin 1, restraining cell migration and invasion,is involved in hepatocellular carcinoma recurrence

Objective

Hepatocellular carcinoma (HCC) is a high‐burden disease. Peroxiredoxin 1 (PRDX1) is a member of the peroxiredoxin family of antioxidant enzymes. The aim of this study was to assess the value of PRDX1 for predicting HCC recurrence after curative resection and to explore the role of PRDX1 in HCC cell migration and invasion.

Methods

Data of patients with HCC who had undergone complete resection between 2002 and 2006 were collected. Immunohistochemical detection of PRDX1 in HCC tissue and adjacent non‐cancerous tissue was conducted. Kaplan–Meier survival estimate and log–rank test were used to assess the relationship between PRDX1 expression and prognostic significance. HCC cell migration and invasion together with the interaction between PRDX1 and ubiquitin C‐terminal hydrolase 37 (UCH37) were studied in vitro.

Results

PRDX1 was expressed at lower levels in HCC tissues than in adjacent non‐cancerous tissues, and PRDX1 was found to be an independent risk factor for disease‐free survival and overall survival. PRDX1 restrained cell migration and invasion in vitro. PRDX1 was found to interact with UCH37 to affect HCC cell migration and invasion.

Conclusion

PRDX1 restrains cell migration and invasion in HCC cell lines and that may be involved in a UCH37‐relevant pathway, suggesting that PRDX1 may be a new marker for HCC recurrence after surgery.     

Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal-regulated kinase/matrix metalloproteinase-9 axis

The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.     

Expression of histone deacetylase 1 and matrix metalloproteinase -2 in hepatoceilular carcinoma and its significance

目的 探讨组蛋白去乙酰化酶1( HDAC1)及基质金属蛋白酶-2(MMP -2)在肝细胞癌(HCC)患者肝组织中的表达及其临床意义.方法 采用免疫组织化学方法检测42例HCC和相应的癌旁组织中HDAC1及MMP -2蛋白的表达,并分析其与临床病理参数的关系.结果 HCC组织中,HDAC1及MMP -2蛋白的表达明显高于癌旁组织(P＜0.01,P＜0.05);HDAC1蛋白的表达与患者性别、年龄、肿瘤大小、AFP浓度、肿瘤分化程度、脉管癌栓无关联(P＞0.05),与肿瘤TNM分期相关联(P＜0.05);MMP -2蛋白的表达与患者性别、年龄、肿瘤大小、AFP浓度、肿瘤分化程度无关联(P＞0.05),与脉管癌栓、肿瘤TNM分期相关联(P＜0.05,P＜0.01 );HCC组织中,HDAC1及MMP -2蛋白的表达呈正相关(P＜0.01).结论 HDAC1及MMP -2在HCC患者肝组织中表达有一定的肿瘤特异性,二者的高表达提示HCC已属晚期.     

芳香烃受体核易位蛋白2基因表达对HCCLM6细胞侵袭和迁移的影响

目的 探讨短发夹(sh)RNA-芳香烃受体核易位蛋白(ARNT)2i慢病毒表达载体对高转移肝癌细胞HCCLM6侵袭和迁移的影响.方法 以ARNT2为目的基因,化学合成4条干扰RNA,与经Mlu Ⅰ和Cla Ⅰ酶切后的pLVTHM载体连接过夜,筛选并鉴定阳性克隆.利用脂质体2000将shRNA-ARNT2i,pCMV-dR8.74,pMD2G共转染293T细胞,获得shRNA-ARNT2i慢病毒颗粒,进一步感染HCCLM6靶细胞,并分为转染shRNA-ARNT2i组、未转染的空白对照组和转染阴性对照序列组.用荧光实时定量PCR和Western blot检测ARNT2基因的干扰效率.应用划痕和Boyden小室法观察ARNT2表达下调对HCCLM6侵袭和迁移能力的影响.用SPSS16.0统计软件对数据进行独立样本t检验和方差分析,其中多组资料两两比较采用LSD法.结果 荧光实时定量PCR检测转染shRNA-ARNT2i组,未转染的空白对照组和转染阴性对照序列组3组ARNT2mRNA相对表达值分别为0.154±0.024、0.860±0.145、1.004±0.009,F=113.14,P<0.01,差异有统计学意义;Western blot显示转染shRNA-ARNT2i组ARNT2蛋白的表达量为16.45±1.6,转染阴性对照序列组ARNT2蛋白的表达量为4_4.56±2.07,两组比较,t=18.58,P<0.01,差异有统计学意义;转染shRNA-ARNT2i组细胞在划痕48 h后基本愈合,而转染阴性对照序列组和未转染的空白对照组划痕依然可见;转染shRNA-ARNT2i组穿膜细胞数为(13.25±1.04)个,而未转染的空白对照组和转染阴性对照序列组的穿膜细胞数分别为(6.50±2.56)个、(6.75±2.05)个,F=29.645,P<0.01,差异有统计学意义.结论 shRNA-ARNT2i载体能显著下调HCCLM6细胞ARNT2基因和蛋白的表达,促进HCCLM6细胞的运动和侵袭.     

组蛋白去乙酰化酶1及基质金属蛋白酶-2在肝细胞癌组织中的表达及意义

目的探讨组蛋白去乙酰化酶1(HDAC1)及基质金属蛋白酶-2(MMP-2)在肝细胞癌(HCC)患者肝组织中的表达及其临床意义。方法采用免疫组织化学方法检测42例HCC和相应的癌旁组织中HDAC1及MMP-2蛋白的表达,并分析其与临床病理参数的关系。结果 HCC组织中,HDAC1及MMP-2蛋白的表达明显高于癌旁组织(P<0.01,P<0.05);HDAC1蛋白的表达与患者性别、年龄、肿瘤大小、AFP浓度、肿瘤分化程度、脉管癌栓无关联(P>0.05),与肿瘤TNM分期相关联(P<0.05);MMP-2蛋白的表达与患者性别、年龄、肿瘤大小、AFP浓度、肿瘤分化程度无关联(P>0.05),与脉管癌栓、肿瘤TNM分期相关联(P<0.05,P<0.01);HCC组织中,HDAC1及MMP-2蛋白的表达呈正相关(P<0.01)。结论 HDAC1及MMP-2在HCC患者肝组织中表达有一定的肿瘤特异性,二者的高表达提示HCC已属晚期。     

人脑胶质瘤转录激活因子3、maspin和基质金属蛋白酶2蛋白表达及意义

目的探讨人脑胶质瘤中转录激活因子3（ATF3）、乳腺丝氨酸蛋白酶抑制因子（maspin）和基质金属蛋白酶2（MMP2）的蛋白表达情况及其与胶质瘤发展的相关性。方法采用免疫组织化学染色法、Western blot法检测100例脑胶质瘤患者和13例健康脑组织中ATF3、maspin和MMP2的蛋白表达相对量。结果ATF3在胶质瘤中的表达（72．0％）明显高于健康脑组织（15．4％）（P〈0．05）,MMP2在胶质瘤中的表达（76．0％）明显高于健康脑组织（7．7％）（P〈0．05）,maspin在胶质瘤中的表达（53．0％）明显低于健康脑组织（100．0％）（P〈0．05）。随胶质瘤病理级别的增高,ATF3和MMP2的表达量逐级增高,而maspin表达量逐级递减。ATF3蛋白表达与maspin蛋白表达呈负相关（r=-0．457,P〈0．01）,ATF3蛋白表达与MMP2蛋白表达呈正相关（r=0．553,P〈0．01）,maspin蛋白表达与MMP2蛋白表达呈负相关（r=-0．551,P〈0．01）。结论ATF3、maspin和MMP2三者间的表达有相关性,ATF3、MMP2高表达及maspin低表达与胶质瘤的发生密切相关,它们可能在胶质瘤发生发展、侵袭转移过程中发挥重要作用。     

siRNA沉默NHE1基因对MHCC97-H肝癌细胞侵袭迁移的影响

目的:探讨RNA干扰沉默NHE1基因后对人肝癌细胞株MHCC97-H细胞侵袭迁移的影响,方法:应用NHE1基因小干扰RNA(smallinterfering RNA,siRNA)转染人肝癌细胞株MHCC97-H细胞,同时设立空白对照组和无关对照组.转染成功后采用RT-PCR和Western blot分别从基因和蛋白水平...     

Chlorotyrosine promotes human aortic smooth muscle cell migration through increasing superoxide anion production and ERK1/2 activation

Chlorotyrosine is an oxidative product of hypochlorous acid and l-tyrosine, and is considered as a biomarker for oxidative stress and cardiovascular disease. However, it is not clear whether chlorotyrosine could directly contribute to vascular pathogenesis. In this study, we investigated the effect and potential mechanisms of chlorotyrosine on human aortic smooth muscle cell (AoSMC) migration. With Boyden chamber and wound healing assays, chlorotyrosine significantly increased AoSMC migration in a concentration- and time-dependent manner. In addition, chlorotyrosine significantly increased the expression of several key molecules related to cell migration including PDGF receptor-B (PDGFR-B), matrix metalloproteinases (MMP-1 and MMP-2) and integrins (alpha3, alphaV, and beta3) in AoSMC at both mRNA and protein levels. Furthermore, chlorotyrosine also increased superoxide anion generation in AoSMC with the fluorescent dye dihydroethidium (DHE) staining. Activation of mitogen-activated protein kinases (MAPKs) was analyzed with Bio-Plex Luminex immunoassay and Western blotting. Chlorotyrosine induced a transient phosphorylation of ERK1/2, but not JNK and p38 MAPKs. Antioxidants including selenomethionine (SeMet) and Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) as well as ERK1/2 inhibitor PD98059 effectively blocked chlorotyrosine-induced AoSMC migration. Thus, these findings demonstrate new biological functions of chlorotyrosine in human SMC migration, which may play a crucial role in the vascular lesion formation.     

Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion

AIM:To evaluate the expression of C-X-C motif chemokine receptor 4(CXCR4)and its signaling cascades,which were previously identified as a key factor for cancer cell progression and metastasis,in cholangiocarcinoma cell lines.METHODS:The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines(RMCCA1 and KKU100)by Western blotting.The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand-12(CXCL12).RESULTS:Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2(ERK1/2)and phosphoinositide 3-kinase(PI3K)and induction of cholangiocarcinoma cell invasion,and displayed high levels of actin polymerization.Addition of CXCR4 inhibitor(AMD3100)abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells.Moreover,treatment with MEK1/2 inhibitor(U0126)or PI3K inhibitor(LY294 002)also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion.CONCLUSION:These results indicated that the activation of CXCR4 and its signaling pathways(MEK1/2 and Akt)are essential for CXCL12-induced cholangiocarcinoma cell invasion.This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.