TROVE2 regulated invasion and migration of hepatocellular carcinoma cells via heparanase

Background:The role of TROVE domain family member 2(TROVE2)has been well-demonstrated in autoimmune diseases;however,its involvement in liver cancer remains unclear.Therefore,this study aimed to explore the biological function and clinical significance of TROVE2 in hepatocellular carcinoma(HCC).Methods:The expression level of TROVE2 was analyzed in HCC and paired adjacent tissue samples using real-time reverse transcription-quantitative polymerase chain reaction.The impact of TROVE2 on migration and invasion in HCC cells was analyzed through Transwell assays and Western blotting.High-throughput transcriptome sequencing and bioinformatics analyses were performed to identify downstream target genes.Back-complementation experiments were employed to verify the influence of downstream proteins on TROVE2-induced invasion and migration of HCC cells.Results:TROVE2 exhibited significant overexpression in liver cancer tissue,correlating with shorter overall survival.Overexpression of TROVE2 facilitated the invasion,metastasis,and epithelial-mesenchymal transition(EMT)process of HCC cells,whereas TROVE2 knockdown restrained migration,invasion,and EMT in these cells.Transcriptome sequencing and bioinformatics analysis identified heparanase(HPSE)as a downstreamtarget protein of TROVE2.Subsequent back-complementation experiments provided evidence that HPSE overexpression promoted TROVE2-mediated prometastasis effects.Moreover,the study revealed that TROVE2 was capable of regulating the EMT pathway through GSK-3βphosphorylation.Conclusions:TROVE2 facilitated the invasion,migration,and EMT process ofHCC cells through phosphorylation of the HPSE/GSK-3βaxis,indicating its significance as an important protein in tumor progression.

Heparanase inhibition leads to improvement in patients with acute gastrointestinal injuries induced by sepsis

BACKGROUND Patients with sepsis are at high risk for acute gastrointestinal injury(AGI),but the diagnosis and treatment of AGI due to sepsis are unsatisfactory.Heparanase(HPA)plays an important role in septic AGI(S-AGI),but its specific mechanism is not completely understood,and few clinical reports are available.AIM To explore the effect and mechanism of HPA inhibition in S-AGI patients.METHODS In our prospective clinical trial,48 patients with S-AGI were randomly assigned to a control group to receive conventional treatment,whereas 47 patients were randomly assigned to an intervention group to receive conventional treatment combined with low molecular weight heparin.AGI grade,sequential organ failure assessment score,acute physiology and chronic health evaluation II score,D-dimer,activated partial thromboplastin time(APTT),anti-Xa factor,interleukin-6,tumour necrosis factor-α,HPA,syndecan-1(SDC-1),LC3B(autophagy marker),intestinal fatty acid binding protein,D-lactate,motilin,gastrin,CD4/CD8,length of intensive care unit(ICU)stay,length of hospital stay and 28-d survival on the 1^(st),3^(rd) and 7^(th) d after treatment were compared.Correlations between HPA and AGI grading as well as LC3B were compared.Receiver operator characteristic(ROC)curves were generated to evaluate the diagnostic value of HPA,intestinal fatty acid binding protein and D-lactate in S-AGI.RESULTS Serum HPA and SCD-1 levels were significantly reduced in the intervention group compared with the control group(P<0.05).In addition,intestinal fatty acid-binding protein,D-lactate,AGI grade,motilin,and gastrin levels and sequential organ failure assessment score were significantly decreased(P<0.05)in the intervention group.However,LC3B,APTT,anti-Xa factor,and CD4/CD8 were significantly increased(P<0.05)in the intervention group.No significant differences in interleukin-6,tumour necrosis factor-α,d-dimer,acute physiology and chronic health evaluation II score,length of ICU stay,length of hospital stay,or 28-d survival were noted between the two groups(P>0.05).Correlation analysis revealed a significant negative correlation between HPA and LC3B and a significant positive correlation between HPA and AGI grade.ROC curve analysis showed that HPA had higher specificity and sensitivity in diagnosis of S-AGI.CONCLUSION HPA has great potential as a diagnostic marker for S-AGI.Inhibition of HPA activity reduces SDC-1 shedding and alleviates S-AGI symptoms.The inhibitory effect of HPA in gastrointestinal protection may be achieved by enhanced autophagy.

人Heparanase基因真核和原核表达载体的构建及融合蛋白的表达

目的 :构建人Heparanase基因的真核、原核表达载体 ,大肠杆菌表达其融合蛋白 .方法 :采用反转录 聚合酶链反应从人肝癌细胞株HepG2cDNA中 ,分别扩增出Heparanase编码基因 ,用限制性内切酶BamHI消化后 ,插入真核表达载体pcDNA3.1中 ,经酶切鉴定与测序证实后 ,连接成包括完整的人Heparanase基因ORF区的真核表达载体 .以亚克隆法构建于原核表达载体pRSET的相应酶切位点 ,转化大肠杆菌BL2 1菌株 ,异丙基 β D硫代半乳糖苷 (IPTG)诱导产生融合蛋白 .结果 :构建的人Heparanase基因表达载体经序列测定证实 ,与GenBank登录结果完全一致 ;双酶切鉴定证实 ,克隆基因正确插入载体pcDNA3.1及pRSET ;SDS PAGE证实融合蛋白表达成功 .结论 :成功构建了人Heparanase基因真核、原核表达载体 ,成功正确表达了

Heparanase、COX-2在肾癌中的表达和肿瘤血管生成的关系

目的:探讨Heparanase、COX-2在肾癌中表达的相关性及与肿瘤血管生成的关系及意义。方法:采用免疫组化PV6000法检测54例肾癌和20例正常肾脏组织中Hpa、COX-2蛋白的表达,以CD34单克隆抗体进行微血管内皮染色,计算微血管密度,分析Hpa、COX-2在肾癌中表达的相关性以及与临床病理特征及微血管密度的关系。结果:Hpa、COX-2在肾癌中的表达均较正常肾脏组织明显增高(P<0.05)。Hpa的表达与肾癌的临床分期、肿瘤大小、远处转移有关(P<0.05),而与患者年龄、性别、肿瘤的分化程度无相关性(P>0.05)。COX-2的表达与肾癌的临床分期、远处转移及分化程度有关(P<0.05),与患者年龄、性别、肿瘤大小无关(P>0.05)。肾癌组织中MVD显著高于正常肾脏组织(P<0.05),高MVD组中Hpa、COX-2表达的阳性率高于低MVD组(P<0.05),Hpa、COX-2阳性表达组中MVD值亦高于阴性表达组(P<0.05)。Hpa、COX-2的表达具有相关性(P<0.05)。结论:Hpa、COX-2可能通过促肿瘤血管生成参与肾癌的恶性生物学行为,并有可能成为判断肾癌预后的指标和靶向治疗的靶点。

Heparanase表达下调对结肠癌细胞增殖和侵袭的影响

目的探讨乙酰肝素酶(Heparanase)对结肠癌细胞生物学功能的影响。方法采用荧光定量PCR检测人结肠癌细胞株SW620、SW480、LoVo和CACO-2中Heparanase的表达水平;设计并合成3条Heparanase特异性的小分子干扰RNA(siRNA1、siRNA2、siRNA3),用于转染4株结肠癌细胞中Heparanase表达水平最高者;荧光定量PCR和Western blotting检验3条siRNA的干扰效果;MTT法检测结肠癌细胞的增殖能力;Transwell小室法检测结肠癌细胞的侵袭能力。结果 SW480细胞中Heparanase的表达水平高于其他3株结肠癌细胞;3条特异性siRNA转染SW480细胞后,均能在蛋白和mRNA水平抑制Heparanase的表达,以siRNA3的效果最佳,且呈剂量依赖性。siRNA3介导的Heparanase基因沉默可呈剂量依赖地降低SW480细胞的增殖能力并减弱侵袭能力(P<0.05)。结论 Heparanase表达下调可导致结肠癌细胞生物学恶性程度降低,提示Heparanase可作为结肠癌靶向治疗的一个重要靶点。

Heparanase-1和CoX-2在嗜铬细胞瘤中的表达及其与血管生成的关系

目的通过检测肝素酶-1(HPA)、环氧化酶-2(CoX-2)、微血管密度(MVD)在良、恶性嗜铬细胞瘤中的表达情况,探讨HPA、CoX-2能否成为一种预判恶性嗜铬细胞瘤的指标,并阐明三者间的相互关系。方法选取1986年10月～2006年8月住院经手术治疗、且有完整的临床、病理和随访资料的嗜铬细胞瘤患者存档石蜡标本38例,其中良性嗜铬细胞瘤21例,恶性嗜铬细胞瘤17例。恶性组中首次手术确诊为嗜铬细胞瘤后随访28～179月,良性组中首次手术确诊为嗜铬细胞瘤后随访93～264月;另取20例因良性肾疾患行肾切除时获取的同侧正常肾上腺组织作为对照组。采用免疫组织化学技术,检测良、恶性嗜铬细胞瘤及对照组中HPA、CoX-2和MVD的表达情况。结果 HPA在恶性组中表达最高,在良性组中表达较低,在对照组中无表达,恶性组与良性组及恶性组与对照组之间HPA的表达有显著性差异(P<0.05)。CoX-2在恶性组中表达最高,良性组中表达较低,对照组中无表达,恶性组与良性组及恶性组与对照组之间CoX-2的表达差异有统计学意义(P<0.05)。MVD在恶性组中呈高表达,在良性组中次之,在对照组中最低,MVD在恶性组中的表达与在良性组和对照组中的表达有显著性差异(P<0.05)。在嗜铬细胞瘤中HPA与CoX-2和MVD的表达两两之间呈正相关。结论 HPA和CoX-2有望成为预判恶性嗜铬细胞瘤的一种指标,并且两者均与肿瘤的血管生成有关。

正、反义Heparanase基因荧光真核表达载体的构建及胰腺癌SW1990细胞转染

目的构建正、反义人肝素酶绿色荧光蛋白真核共表达载体,并转染人胰腺癌SW 1990细胞.方法用EcoR I从pcDNA3-Hpa质粒上切下约1.7 kb的人肝素酶全长cDNA片段,然后连入pIRES2-EGFP质粒的EcoR I酶切位点上,经BamH I酶切鉴定出正义和反义表达载体,并进一步测序确认.用脂质体法将肝素酶正、反义真核表达载体转染人胰腺癌SW 1990细胞,24 h后荧光倒置显微镜下检测转染结果.结果经BamH I酶切后,正义质粒形成5.3 kb和1.7 kb两条DNA片段,反义重组质粒为6.5 kb和0.5 kb两条DNA片段,与理论计算值一致;测序结果进一步确认了方向的正确性.转染成功的胰腺癌细胞在倒置荧光显微镜下呈绿色荧光.结论成功构建了人肝素酶正、反义真核表达载体,并成功转染人胰腺癌细胞株SW 1990,为进一步研究正、反义肝素酶基因转染对胰腺癌细胞的转移能力的影响奠定基础.

Heparanase与bFGF在膀胱移行细胞癌中的表达研究

目的探讨类肝素酶(heparanase,Hpa)与碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)在膀胱移行细胞癌(transitional cell carcinoma of the bladder,TCCB)发生、发展中的作用及意义。方法应用免疫组织化学S-P法,检测69例BTCC和9例正常膀胱组织中Hpa和bFGF的表达。结果膀胱癌中,Hpa阳性表达率为42.03%;bFGF阳性表达率为44.93%;Hpa与bFGF共表达阳性率为31.89%;9例正常膀胱黏膜中均未见Hpa和bFGF阳性表达。Hpa与bFGF表达均随着膀胱癌病理分级和临床分期的升高而增强(P<0.05)。结论Hpa与bFGF可能作为预测膀胱移行细胞癌进展的指标及肿瘤治疗靶点,并有可能成为一个有效的预后指标。

Heparanase对肝癌细胞生物学行为的影响

目的探讨乙酰肝素酶(Heparanase)对肝癌细胞生物学功能的影响。方法利用荧光定量PCR检测人肝癌细胞HepG2、Huh-7、Bel-7402、MHCC-97H以及SMMC-7721中Heparanase的表达水平;设计及合成3条Heparanase特异性小分子干扰RNA(siRNA)并用于转染Heparanase表达高的一株人肝癌细胞,通过荧光定量PCR和Western blotting检验干扰效率;MTT法检测细胞增殖能力;Transwell小室法检测细胞的侵袭能力;Annexin V/PI染色法检测细胞凋亡。结果 MHCC-97H细胞中的Heparanase表达水平显著高于其余4株肝癌细胞(P<0.05);siRNA3能瞬时下调肝癌细胞的Heparanase水平;siRNA介导的Heparanase基因沉默使肝癌细胞的增殖速度放慢,侵袭能力减弱,凋亡率升高,差异具有统计学意义(P<0.05)。结论在肝癌细胞中,Heparanase基因的表达下调导致其生物学恶性程度降低,提示Heparanase基因可作为肝癌靶向治疗的一个重要靶点。

GST-His-Heparinase Ⅰ双标签融合蛋白的原核表达与纯化

以pET15b-HepⅠ为模板,通过PCR技术扩增出上游合有6×His标签的HepⅠ基因序列,克隆至表达载体pGEX-4T-1。测序鉴定后,将重组表达质粒pGEX-His-HepⅠ转入E.coli BL21(DE3)感受态细菌,经IPTG诱导表达。表达产物可溶部分用GSTrap FF和HisTrap HP柱两步亲和纯化,所得产物经SDS-PAGE检测,在66 kDa和43 kDa处显示特异条带,分别与GST-His-HepⅠ和His-HepⅠ融合蛋白预期分子量相符;最终His-HepⅠ融合蛋白的比酶活为86.45 IU/mg,纯度高达99%,与仅一步亲和纯化得到的GST-His-HepⅠ融合蛋白相比,进一步提高了纯化后重组肝素酶的纯度。本研究为制备高纯度的HepⅠ提供了一种方法,对制备高安全性的LMwH和解析HepⅠ晶体结构具有重要意义。

RNA干扰Heparanase基因表达对胆囊癌侵袭性影响的体外研究

目的:利用RNA干扰技术沉默人胆囊癌GBC-SD细胞中乙酰肝素酶(heparanase,HPA)基因的表达,探讨抑制HPA基因后对GBC-SD细胞侵袭性的影响。方法:构建靶向人HPA基因miRNA重组质粒,采用脂质体转染法将质粒转染入人胆囊癌GBC-SD细胞,RT-PCR检测转染前后HPA mRNA表达情况,选出对HPA基因沉默效果最佳的质粒。通过划痕损伤试验和Transwell侵袭试验检测干扰沉默HPA表达后对人胆囊癌GBC-SD细胞侵袭性的影响。结果:质粒pcDNA6.2-GW/EmGFP-miR-HPA-2沉默人胆囊癌GBC-SD细胞HPA mRNA表达的效果最好。划痕损伤试验中胆囊癌GBC-SD细胞的迁移距离在12 h、24 h pcDNA6.2-GW/EmGFP-miR-HPA-2组均低于阴性对照组和空白组(P<0.05～P<0.01)。pcDNA6.2-GW/EmGFP-miR-HPA-2组中穿过Transwell小室基质的胆囊癌GBC-SD细胞数均明显低于阴性对照组和空白组(P<0.01)。结论:靶向人HPA基因miRNA重组质粒能有效抑制HPA基因的表达,抑制人胆囊癌GBC-SD细胞的侵袭能力。

Helicobacter pylori promotes invasion and metastasis of gastric cancer by enhancing heparanase expression

AIM To detect the mechanisms of Helicobacter pylori(H. pylori) infection in the invasion and metastasis of gastric cancer(GC).METHODS Specimens from 99 patients with GC were collected. The correlation among H. pylori infection, heparanase(HPA) and mitogen-activated protein kinase(MAPK) expression, which was determined by immunohistochemistry, and the clinical features of GC was analysed using SPSS 22.0. Overall survival(OS) and relapse-free survival(RFS) of GC patients were estimated by the KaplanMeier method. Independent and multiple factors of HPA and MAPK with prognosis were determined with COX proportional hazards models. HPA and MAPK expression in MKN-45 cells infected with H. pylori was analysed using Western blot. RESULTS H. pylori infection was observed in 70 of 99 patients with GC(70.7%), which was significantly higher than that in healthy controls. H. pylori infection was related to lymph metastasis and expression of HPA and MAPK(P < 0.05); HPA expression was relevant to MAPK expression(P = 0.024). HPA and MAPK expression in MKN-45 cells was significantly upregulated following H. pylori infection and peaked at 24 h and 60 min, before decreasing(P < 0.05). SB203580, an inhibitor of MAPK, significantly decreased HPA expression. HPA was related to lymph metastasis and invasive depth. HPA positive GC cases and H. pylori positive GC cases showed poorer prognosis than HPA negative cases(P < 0.05). COX models showed that the prognosis of GC was connected with HPA expression, lymph metastasis, tissue differentiation, and invasive depth. CONCLUSION H. pylori may promote the invasion and metastasis of GC by increasing HPA expression that may associate with MAPK activation, thus causing a poorer prognosis of GC.

Heparanase and hepatocellular carcinoma:Promoter or inhibitor?

Heparan sulphate proteoglycans (HSPGs) consist of a core protein and several heparan sulphate (HS) side chains covalently linked. HS also binds a great deal of growth factors, chemokines, cytokines and enzymes to the extracellular matrix and cell surface. Heparanase can specially cleave HS side chains from HSPGs. There are a lot of conflicting reports about the role of heparanase in hepatocellular carcinoma (HCC). Heparanase is involved in hepatitis B virus infection and hepatitis C virus infection, the activation of signal pathways, metastasis and apoptosis of HCC. Heparanase is synthesized as an inactive precursor within late endosomes and lysosomes. Then heparanase undergoes proteolytic cleavage to form an active enzyme in lysosomes. Active heparanase translocates to the nucleus, cell surface or extracellular matrix. Different locations of heparanase may exert different activities on tumor progression. Furthermore, enzymatic activities and non-enzymatic activities of heparanase may play different roles during HCC development. The expression level of heparanase may also contribute to the discrepant effects of heparanase. Growth promoting as well as growth inhibiting sequences are contained within the tumor cell surface heparan sulfate. Degrading different HSPGs by heparanase may play different roles in HCC. Systemic studies examining the processing, expression, localization and function of heparanase should shed a light on the role of heparanase in HCC.

Combined Detection of Serum Matrix Metalloproteinase 9,Acetyl Heparinase and Cathepsin L in Diagnosis of Ovarian Cancer

Objective： To investigate the clinic values of combining test of serum matrix metalloproteinase 9 （MMP-9）, acetyl heparinase （Hpa） and Cathepsin L （CL） in diagnosis of ovarian cancer. Methods： Serum levels of MMP-9, Hpa and CL were detected in a total of 418 cases, including 217 cases with ovarian malignant tumor, 100 cases with ovarian benign tumor and 101 healthy controls, by using enzyme-linked immunosorbent assay （ELISA）. Their correlation with clinicopathologic feature of ovarian malignant tumor was analyzed and their diagnosis performance was evaluated by receiver operating characteristic （ROC）. The combined diagnosis model was established by logistic regression analysis. Results： The serum levels of MMP-9, Hpa and CL were significantly higher in patients with ovarian malignant tumor than in benign tumor and healthy control, the serum levels of CL and Hpa were higher in epithelial cancer than in non-epithelial tumor, and MMP-9, Hpa and CL were elevated in low grade and advanced stage compared to high grade and early stage. The sensitivity for diagnosis of ovarian malignant tumor from high to low was CL, Hpa and MMP-9, and the specificity was MMP-9, CL and Hpa. The united diagnosis model was established and showed the sensitivity and specificity of combined detection were 84.6% and 82.1%, respectively, which were significantly higher than a single tumor marker. Conclusion： Serum MMP-9, Hpa and CL were correlated with ovarian malignant tumor and the combined detection of which may be valuable for clinical diagnosis of ovarian malignant tumor.

Targeted Silencing of Heparanase Gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pGenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pGenesil） transfected group and expression vector（pGenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Transfection of Antisense Oligodeoxynucleotide Inhibits Heparanase Gene Expression and Invasive Ability of Human Pancreatic Cancer Cell in vitro

Extracellular matrix （ECM） degradation is an essential step that allows tumor cells to penetrate a tissue barrier and become metastatic. Heparanase （HPSE） is an endoglycosidase that specifically degrades heparin sulfate proteoglycans （HSPG）, a chief component of ECM, HPSE is not expressed in normal epithelial cells but can be detected in a variety of human carcinomas including pancreatic cancer. In the present study, human pancreatic cancer cell line Panc-1 was transfected with HPSE antisense oligodeoxynucleotide （AS-ODN） in vitro, then the inhibitory effect of ASODN on HPSE gene expression and invasive ability of Panc-1 cells in vitro was examined. The HPSE mRNA and protein expression of Panc-1 cells transfected with AS-ODN was significantly inhibited. However, there were no marked inhibitory effects in Panc-1 cells treated with nonsense oligodeoxynucleotide （NS-ODN）. Moreover, a modified Boyden chamber assay demonstrated that transfection with HPSE AS-ODN significantly inhibited invasive potential of Panc-1 cells in vitro after AS-ODN transfection. This suggests that HPSE AS-ODN may contribute to the inhibition of HPSE mRNA and protein expression, and results in a decrease of the invasive ability of Panc-1 in vitro.

Expression and significance of heparanase and nm23-Hl in hepatocellular carcinoma

AIM: To explore the relation between heparanase (HPA)and nm23-H1 in hepatocellular carcinoma (HCC), and whether they could be used as valuable markers in predicting post-operative metastasis and recurrence of HCC.METHODS: Reverse transcription-polymerase chain reaction and immunohistochemistry (S-P method) were used to measure the expressions of HPA mRNA and nm23-H1protein in primary tumor tissue and paracancerous tissue of 33 cases of HCC. Paracancerous tissues of 9 cases of benign liver tumor were used as normal controls. The results were analyzed in combination with the results of clinicopathological examination and follow-up.RESULTS: The positive expression of HPA gene was significantly higher in primary tumor tissues of HCC (48.5%,L6/33) as compared to the paracancerous tissues of HCC and normal controls (3.03%, 1/33) (P＜0.01). HPA expression was not related with the size of tumor, envelope formation, AFP level, HBsAg state and cirrhosis of liver.The positive rates of HPA mRNA in the group with high tendency to metastasis or recurrence and in the group with metastasis or recurrence during the follow-up were significantly higher than those in the group with low tendency to metastasis or recurrence (62.5% vs 37.5%,P＜0.05) and in the group without metastasis or recurrence (78.6% vs 21.4%, P＜0.01). The poorly differentiated tumor and tumor of TNM stages Ⅲ-Ⅳ had a higher positive rate of HPA gene expression than the well differentiated tumor and tumor of TNM stages Ⅰ-Ⅱ (66.7% vs 33.3%, P＜0.05). The positive expression rate of nm23-H1 protein in HCC tissue was significantly lower than that in corresponding non-cancerous or normal liver tissue (45.5, 72.7, 88.9%, P＜0.05). nm23-H1 expression was not related with the size of tumor, envelope formation,AFP level, HBsAg state, cirrhosis of liver, Edmondson grade,and TNM stage (P＞0.05). The positive rates of nm23-H1 in the group with high tendency to metastasis and recurrence and in patients with metastasis or recurrence during the follow-up were obviously higher than those in the group with low tendency to metastasis and recurrence (P = 0.018) and in the patients without metastasis and recurrence (P = 0.024); but no significant difference was found between HPA positive and negative groups(P = 0.082). According to the results of follow-up, the rate of accuracy in predicting metastasis of positive HPA,negative nm23-H1 an1378-12-381d combination of positive HPA with negative nm23-H1 was 78.6% (11/114), 68.8% (11/16)and 88.9% (8/9), respectively.CONCLUSION: Expression of HPA and/or nm23-H1 is related with metastasis and recurrence of HCC. Detection of the expression rate of HPA and nm23-H1 may help increase the accuracy in predicting post-operative metastasis and recurrence of HCC.

Tumor metastasis and the reciprocal regulation of heparanase gene expression by nuclear factor kappa B in human gastric carcinoma tissue

AIM: To investigate whether NF-κB is activated in human gastric carcinoma tissues and, if so, to study whether there is any correlation between NF-κB activity and heparanase expression in gastric carcinoma.METHODS: NF-κB activation was assayed by immunohistochemical staining in formalin-fixed, paraffin-embedded specimens from 45 gastric carcinoma patients. Electrophoretic mobility shift assay (EMSA) method was used for nuclear protein from these fresh tissue specimens. Heparanase gene expression was quantified using quantitative RT-PCR.RESULTS: The nuclear translocation of RelA (marker of NF-κB activation) was significantly higher in tumor cells compared to adjacent and normal epithelial cells [(41.3±3.52)% vs (0.38±0.22) %, t= 10.993, P= 0.000＜0.05; (41.3±3.52)%vs (0±0.31)%, t = 11.484, P = 0.000＜0.05]. NF-κB activation was correlated with tumor invasion-related clinicopathological features such as lymphatic invasion,pathological stage, and depth of invasion (Z = 2.148,P= 0.032＜0.05; χ2 = 8.758, P= 0.033＜0.05; χ2 = 18.531,P = 0.006＜0.05). NF-κB activation was significantly correlated with expression of heparanase gene (r= 0.194,P= 0.046＜0.05).CONCLUSION: NF-κB RelA (p65) activation was related with increased heparanase gene expression and correlated with poor clinicopathological characteristics in gastric cancers. This suggests NF-κB as a major controller of the metastatic phenotype through its reciprocal regulation of some metastasis-related genes.

Stable knockdown of heparanase expression in gastric cancer cells in vitro

AIM:To develop short hairpin RNA(shRNA)against heparanase,and to determine its effects on heparanase expression and the malignant characteristics of gastric cancer cells. METHODS:Heparanase-specific shRNA was constructed and transferred into cultured the gastric cancer cell line SGC-7901.Stable subclonal cells were screened by G418 selection.Heparanase expression was measured by reverse transcriptase-polymerase chain reaction(RT-PCR),real-time quantitative PCR and Western blotting.Cell proliferation was detected by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)colorimetry and colony formation assay. The in vitro invasiveness and metastasis of cancer cells were measured by cell adhesion assay,wound healingassay and matrigel invasion assay.The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells. RESULTS:Stable transfection of heparanase-specific shRNA,but not of scrambled shRNA and mock vector,resulted in reduced mRNA and protein levels of heparanase.The shRNA-mediated knockdown of heparanase did not affect the cellular proliferation of SGC-7901 cells.However,the in vitro invasiveness and metastasis of cancer cells were decreased after knockdown of heparanase.Moreover,transfection of heparanase-specific shRNA decreased the in vitro angiogenesis capabilities of SGC-7901 cells. CONCLUSION:Stable knockdown of heparanase can efficiently decrease the invasiveness,metastasis and angiogenesis of human gastric cancer cells.In contrast,stable knockdown of heparanase does not affect the cell proliferation.

heparanase通过调控EMT促进人膀胱移行细胞癌细胞T24迁移及侵袭的作用研究

目的探讨乙酰肝素酶(heparanase)是否通过上皮-间充质转化(EMT)影响膀胱移行细胞癌细胞的侵袭及迁移能力。方法利用荧光定量PCR来检测人膀胱移行细胞癌细胞T24、EJ、MGH-U1以及BIU-87中heparanase的表达水平;设计并合成靶向heparanase的特异性shRNA,通过脂质体转染法转染heparanase表达最高的膀胱移行细胞癌细胞T24以构建稳定低表达heparanase细胞株,通过Western blot法检测和定量PCR来验证shRNA的干扰效率;通过transwell法检测干扰后细胞的迁移及侵袭能力,Western blot法检测EMT相关指标E-cadherin和N-cadherin以及其上游信号分子Snail和WNT-5a的变化。结果 heparanase-shRNA转染后,能够有效抑制T24细胞中heparanase的表达;Transwell法结果显示,heparanase干扰组细胞侵袭及迁移能力显著低于阴性对照组(P<0.001)。Western blot法检测结果发现,heparanase干扰组细胞的E-cadherin表达增加,N-cadherin的表达降低;此外,heparanase干扰组细胞的Snail以及WNT-5a表达较对照组显著下降。结论运用RNA干扰技术能够有效沉默T24细胞的heparanase基因,并诱导其迁移侵袭能力的下降,其可能的机制是通过调节EMT的上游信号分子Snail、WNT-5a的表达来调控EMT,从而影响膀胱移行细胞癌细胞的迁移及侵袭。提示heparanases可能在膀胱移行癌T24细胞的发生发展中起重要作用,抑制heparanase的表达可能成为一种治疗膀胱移行细胞癌的新方法。