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ABSTRACT Objective To investigate the possible genes that may be related to the mechanisms that modulate heparanase-1. Methods The analysis was conducted at Universidade Federal de São Paulo, on the data provided by: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database and the softwares cBioPortal and Ingenuity Pathway Analysis. Results Using messenger RNA expression pattern of different molecular subtypes of breast cancer, we proposed that heparinase-1 was co-related with its progression. In addition, genes that were analyzed presented co-expression with heparanase-1. The results that showed that heparanase-1 co-expressed with phosphoinositide 3-kinase adapter protein 1, sialic acid-binding immunoglobulin-like lectin 7, and leukocyte-associated immunoglobulin-like receptor 1 are directed related with immune system evasion during breast cancer progression. Furthermore, cathepsin L was co-expressed with heparanase-1 and transformed inactive heparanase-1 form into active heparanase-1, triggering extracellular matrix remodeling, which contributes to enhanced tumor-host interaction of the tumor. Conclusion The signaling pathway analysis using bioinformatics tools gives supporting evidence of possible mechanisms related to breast cancer development. Evasion genes of the immune system co-expressed with heparanase-1, a enzyme related with tumor progression.
RESUMO Objetivo Investigar os genes que podem estar relacionados aos mecanismos que modulam a heparanase-1. Métodos A análise foi realizada na Universidade Federal de São Paulo, utilizando dados fornecidos por: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database e os softwares cBioPortal e Ingenuity Pathway Analysis. Resultados Usando o perfil de expressão de RNA mensageiro de diferentes subtipos moleculares de câncer de mama, propusemos que a heparanase-1 esteve correlacionada com a progressão tumoral. Além disso, os genes analisados apresentaram coexpressão com heparanase-1. Os resultados mostraram que a heparanase-1 coexpressa com proteína adaptadora 1 da fosfoinositídeo 3-quinase, lectina 7 tipo Ig de ligação ao ácido siálico e receptor 1 do tipo imunoglobulina associado a leucócitos, estes genes estão diretamente relacionados à evasão do sistema imune durante a progressão do câncer de mama. Além disso, a catepsina L foi coexpressa com a heparanase-1 e transformou a forma inativa da heparanase-1 em heparanase-1 ativa, desencadeando o remodelamento da matriz extracelular, o que contribuiu para a interação do tumor com o ambiente tumoral. Conclusão A análise utilizando bioinformática fornece evidências de possíveis mecanismos relacionados ao desenvolvimento do câncer de mama. Genes de evasão do sistema imune foram coexpressos com a heparanase-1, uma enzima relacionada à progressão tumoral.
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Humans , Breast Neoplasms/genetics , Glucuronidase/genetics , Computer SimulationABSTRACT
OBJECTIVE@#To explore the effect of heparanase (HPSE) on apoptosis of microvascular endothelial cells (MVECs) and trans-endothelial migration of hepatocellular carcinoma (HCC) cells.@*METHODS@#A HCC cell line with high HPSE expression was selected by real-time quantitative PCR (qRT-PCR) and Western blotting and transefected with a lentiviral vector containing an interfering RNA sequence of HPSE. Transwell migration assay was performed to detect the trans-endothelial migration (TEM) rate of the transfected HCC cells across human umbilical vein endothelial cells (HUVECs). In a Transwell indirect co-culture system, the effect of HPSE silencing in the HCC cells was determined on apoptosis of HUVECs . A nude mouse model of HCC was used to verify the effect of HPSE on apoptosis of MVECs and liver metastasis of the tumor.@*RESULTS@#HCCLM3 cell line highly expressing HPSE was selected for the experiment. Transfection of the HCC cells with the lentiviral vector for HPSE interference the HCC cells resulted in significantly lowered TEM rate as compared with the cells transfected with the control vector ( < 0.01). In the indirect co-culture system, the survival rate of HUVECs co-cultured with HCCLM3 cells with HPSE interference was significantly higher and their apoptotic index was significantly lower than those in the control group ( < 0.05). Ultrastructural observation showed no obvious apoptosis of HUVECs co-cultured with HCCLM3 cells with HPSE interference but revealed obvious apoptotic changes in the control group. In the animal experiment, the tumor formation rate in the liver was 100% (6/6) in the control group, significantly higher than that in RNAi group (33.3%, 2/6) ( < 0.05). Under optical microscope, necrosis and apoptosis of the MVECs was detected in the liver of the control mice, while the endothelial cells remained almost intact in RNAi group.@*CONCLUSIONS@#HPSE promotes the metastasis of HCC cells by inducing apoptosis of MVECs.
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Animals , Humans , Mice , Apoptosis , Carcinoma, Hepatocellular , Cell Line, Tumor , Cell Movement , Cell Proliferation , Endothelial Cells , Gene Expression Regulation, Neoplastic , Glucuronidase , Liver NeoplasmsABSTRACT
Objective To investigate the expression characteristics of heparinase (Hpa) and angiogenesis in human carotid atherosclerotic plaques. Methods Fifty-five consecutive patients with atherosclerotic stenosis in internal carotid artery system treated with carotid endarterectomy (atherosclerosis group) at the Department of Neurosurgery,Fuxing Hospital,Capital Medical University from December 2014 to December 2017 were enrolled retrospectively. They were confirmed by imaging methods,such as vascular ultrasound, CT angiography and/or DSA. According to the atherosclerotic histopathological classification method of American Heart Association, the well-defined advanced atherosclerotic plaques (types IV- VI) were screened as carotid atherosclerotic plaque specimens (ra =73). According to the morphological classification criteria,73carotid atherosclerotic plaque specimens were divided into vulnerable plaques (n =42) and metastatic plaques (n =31). Autopsy was performed at the Department of Pathology, Peking University Medical School at the same time,and 15 patients without atherosclerotic lesions were identified as non-atherosclerotic group (15 normal arterial specimens). Immunohistochemistry and immunofluorescence staining were used to analyze the expression characteristics of Hpa and neovascularization in carotid atherosclerotic plaque. Results (1) There was no Hpa expression in normal arterial tissue; in the carotid atherosclerotic plaque,the positive expression rate of Hpa was 64.4% (47/73). (2) Compared with the metastatic plaques, the positive expression rate of Hpa in the vulnerable plaques increased (92. 9% [39/42] vs. 25. 8% [8/ 31] ,x∗= 34.968,P<0.01). (3) The expression of neovascularization was positive in carotid atherosclerotic plaques. Compared with the metastatic plaques,the positive expression rate of neovascularization in the vulnerable plaques was higher (95. 2% [40/42] vs. 22. 6% [7/31] # =41. 060,P <0. 01). (4) In carotid atherosclerotic plaques, Hpa and neovascularization expression was mainly located in the fibrous cap and shoulders of the plaques. Conclusions Hpa has a certain role in the progression of advanced atherosclerotic lesions. Hpa and angiogenesis expression in carotid atherosclerotic plaques has a co-regionality.
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Objective To investigate the expression of heparanase (HPA) and NF-E2 associated factor (NRF2) in laryngeal squamous cell carcinoma.Methods We collected 81 cases of laryngeal squamous cell carcinoma in our hospital from February 2015 to February 2017 as group A,and then selected 77 cases of polyp of vocal cord pathological specimens as group B.The expression of HPA was detected by immunohistochemistry,and the expression of NRF2 was detected by western blot.Results The positive expression rate of HPA and the expression of NRF in the group A and B were 81.48％,19.48％ and 0.844±0.113,0.202±0.094,the difference was statistically significant (P＜ 0.05).The positive expression rates of HPA in patients with lymph node metastasis and TNM stage Ⅲ to Ⅳ were 93.02％ and 94.87％,which were significantly higher than those without lymph node metastasis and stage Ⅰ to Ⅱ (P＜0.05).The positive expression rates of HPA in patients with low and middle differentiation were 93.75％ and 100.00％,which were significantly higher than those with high differentiation (P＜0.05).The expression of NRF2 in patients with lymph node metastasis and TNM stage Ⅲ to Ⅳ were 0.901± 0.122 and 0.885 ± 0.105,which significantly higher than those without lymph node metastasis and stage Ⅰ to Ⅱ (P＜0.05).The expression of NRF2 in patients with moderate and low differentiation were 0.854± 0.101 and 0.878 ± 0.099,which were significantly higher than patients with stage Ⅰ to Ⅱ (P＜0.05).Conclusion The expression of HPA and NRF2 in laryngeal squamous cell carcinoma are significantly increased,which are related to lymph node metastasis,TNM stage and pathological grade.
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Abstract Heparanase activity is involved in cancer growth and development in humans and single nucleotide polymorphisms (SNPs) in the heparanase gene (HPSE) have been shown to be associated with tumors. In this study, we investigated whether SNPs in HPSE were a risk factor for hepatocellular carcinoma (HCC) by undertaking a comprehensive haplotype-tagging, case-control study. For this, six haplotype-tagging SNPs (htSNPs) in HPSE were genotyped in 400 HCC patients and 480 controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. A log-additive model revealed significant correlations between the HPSE polymorphisms rs12331678 and rs12503843 and the risk of HCC in the overall samples (p = 0.0046 and p = 0.0055). When the analysis was stratified based on hepatitis B virus (HBV) carrier status, significant interactions between rs12331678 and rs12503843 and HBV were observed. Conditional logistic regression analysis for the independent effect of one significant SNP suggested that rs12331678 or rs12503843 contributed an independent effect to the significant association with the risk of HCC, respectively. Our findings suggest that the SNPs rs12331678 and rs12503843 are HCC risk factors, although the potential functional roles of these two SNPs remain to be fully elucidated.
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Objective To investigate the effect of unfractionated heparin on the expression of serum and liver tissue heparanase (HPA) in mice with liver injury induced by sepsis. Methods Forty-eight healthy male C57BL/6 mice aged 6-8 weeks were divided into groups according to random number table method. Twenty-four septic mice models (CLP group) were established by cecal ligation and puncture (CLP); the other 24 mice underwent sham operation (sham group), only laparotomy and abdominal closure were performed without ligation. Twelve mice in sham group and CLP group received heparin pretreatment (sham+UFH group, CLP+UFH group), and 8 U heparin unfractionated heparin (diluted to 200 μL) was injected into the tail vein of the mice at 30 minutes and 12 hours after operation respectively. The other 12 mice were injected with the same amount of normal saline. The serum and liver tissues of mice were collected at 4 and 24 hours after CLP. The levels of serum HPA, interleukin (IL-6, IL-1β), tumor necrosis factor-α (TNF-α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by enzyme linked immunosorbent assay (ELISA). The pathological changes of liver tissue were observed with hematoxylin eosin (HE) staining. The expression of HPA in liver tissue was detected by immunohistochemistry. Results Compared with the sham group, the levels of serum HPA, IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were increased significantly, and increased further over time. The histopathology examination was performed, and abnormal structure, inflammatory cell infiltration, liver cell necrosis could be found in the tissue. The expression level of HPA in liver tissue was detected by immunohistochemistry, which was increased after CLP. This indicated that the animal model of sepsis was successfully prepared. Compared with CLP group, serum HPA, inflammatory factors and transaminase levels were significantly decreased at 4 hours after operation in group CLP+UFH [HPA (ng/L): 76.72±2.75 vs. 101.55±7.54, IL-6 (ng/L): 51.16±5.68 vs. 63.89±3.26, IL-1β (ng/L): 31.53±2.90 vs. 40.87±2.88,TNF-α (ng/L): 171.76±5.60 vs. 194.62±14.13, ALT (μg/L): 0.26±0.09 vs. 0.62±0.17, AST (μg/L): 1.03±0.22 vs. 1.45±0.08, all P < 0.05]. At 24 hours, it was significantly higher than that of 4 hours, but they were significantly lower than those in CLP group [HPA (ng/L): 125.30±7.80 vs. 302.50±17.81, IL-6 (ng/L): 81.16±4.54 vs. 176.56±5.45, IL-1β (ng/L): 61.13±2.80 vs. 113.73±3.96, TNF-α (ng/L): 328.47±10.79 vs. 599.62±10.20, ALT (μg/L): 0.38±0.17 vs. 0.91±0.26, AST (μg/L): 1.16±0.15 vs. 1.88±0.08, all P < 0.05]. It was shown by HE staining that the edema of liver tissue decreased and inflammatory cell infiltration decreased. It was shown by immunohistochemistry that the expression level of HPA in liver tissue was significantly decreased [A value (×10-3): 2.49±0.93 vs. 6.05±1.22 at 4 hours, 1.86±0.77 vs. 7.55±0.35 at 24 hours, both P < 0.05]. There was no significant difference in indexes between the sham+UFH group and the sham group. Conclusions The expression of HPA was significantly increased during sepsis in mice. Unfractionated heparin may mitigate liver injury by inhibiting HPA.
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Objective To investigate the role of heparanase (HPSE) in regulating the liver regeneration after major hepatectomy in rats. Methods The HPSE small interfering RNA (siRNA) was designed and synthesized, and was transfected into the rats after 70% hepatectomy by in wwo-jetPE--Gal vector. Then immunohistochemistry was performed to determine the expression of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). Western blotting analysis was performed to detect the expression of matrix metalloprotein (MMP)-2 and MMP-9 protein expression. Immunostaining for VIII factor was done to observe the microvessels, and the microvessel density (MVD) was calculated. Results Compared with control group, the expressions of HGF, VEGF, MMP-2 and MMP-9 were significantly inhibited in HPSE siRNA group, and theMVD was significantly decreased (P<0. 01). Conclusion HPSE may regulate liver regeneration via promoting HGF release in ECM, inducing hepatocyte proliferation, increasing VEGF expression to accelerate angiogenesis and upregulating the expression of MMP-2 and MMP-9.
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Objective To measure HPSE and VEGF-C expression in pancreatic cancer tissues and to observe the effect of small interfering RNA targeting heparanase gene on VEGF-C expression and malignant biological behavior of pancreatic cancer cell lines BxPC-3.Methods We measured the relative mRNA levels of HPSE and VEGF-C in 34 pancreatic ductal cell adenocarcinoma(PDAC) specimens by RT-qPCR.The recombinant plasmid GV230/HPSE was constructed.BxPC-3 was transiently transfected with GV230/HPSE and HPSE-siRNA.The expression levels of HPSE and VEGF-C were measured by performing florescent real-time quantitative PCR (RT-qPCR) and immunoblotting.The invasion and migration potential of treated BxPC-3 was evaluated by transwell invasion assay and Scratch-wound assay.Results Spearman rank correlation analysis indicated a positive correlation between HPSE and VEGF-C (r =0.812,P ＜0.01)in pancreatic cancer tissues.HPSE and VEGF-C overexpression was correlated with tumor differentiation,lymph node metastasis and TNM stage(all P ＜0.01).RT-qPCR and Western blot showed high expression of HPSE mRNA and VEGF-C mRNA in GV230/HPSE group than that in control group,compared to low expression of HPSE mRNA and VEGF-C mRNA in siRNA group (all P ＜ 0.01).Invasion chamber assay and Scratch-wound assay showed higher invasion in GV230/HPSE group than that in control group,while lower invasion in siRNA group (all P ＜ 0.01).Conclusions Joint detection of HPSE and VEGF-C may be of significance in predicting prognosis of patients with pancreatic cancer.HPSE regulates the expression of VEGF-C and facilitates invasion and migration of BxPC-3 in vitro.
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Objective To explore the inhibitory effects of sulfated oligosaccharides PI-88 on the heparanase protein expression of human esophageal squamous cancer cell (ESCC) line TE-13, and to explore the effects of growth, angiogenesis and heparanase protein expression on ESCC xenografts of nude mice. Methods TE-13 cells were cultured and divided into three groups:group A (control group), group B (15 mg/L PI-88) and group C (30 mg/L PI-88). Heparanase protein expression of TE-13 cells was measured by Western blot assay after being cultured for 36 h. The ESCC suspension was injected subcutaneously in 10 BALB/c/nu mice to build up ESCC xenograft model. The model mice were divided randomly into observation group and control group (5 mice per group). The mice in observation group received 40 mg/(kg·d) PI-88. The mice in control group only received the same volume of saline at the same time. Both PI-88 and saline were daily administrated for 14 days. Every 2 days,the volume of xeongrafts were measured and the mice were executed at the 14th day. CD34 immunohistochemical staining was used to detect the micro vessel density (MVD) of xenografts. Western blot assay and immunohistochemical staining were used to detect the heparanase protein expression of xenografts. Results The heparanase protein expressions of TE-13 cells were significantly decreased in group B and group C than those of group A (P<0.001), with a kind of PI-88 dose-dependent manner. The volume, MVD and heparanase protein expression of xenografts were significantly lower in observation group than those of control group (P<0.05). Conclusion The heparanase protein expression in TE-13 cells can be inhibited by PI-88 in vitro and vivo. Furthermore, the growth and angiogenesis of ESCC xenografts were also inhibited by PI-88.
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Objective: To investigate the effects of sodium cantharidinate and vitamin B6 (SCAVB6) on cell proliferation, apoptosis and invasion of hepatic cancer cell line SMMC-7721 as well as the expression of heparanase (HPA). Methods: After treatment with 0.5, 1.0, 1.5 and 2.0 μg/mL SCAVB6 for 24, 48 and 72 h, the cell proliferation was detected by MTT assay. The cell apoptosis, invasion ability and the expression levels of HPA mRNA and protein of SMMC-7721 cells after treatment with 0.5, 1.0, 1.5 and 2.0 μg/mL SCAVB6 for 48 h were examined by flow cytometry (FCM), Transwell cell invasion assay, RT-PCR and Western blotting, respectively. Results: SCAVB6 could inhibit the proliferation of SMMC-7721 cells in a dose- and time-dependent manner (P < 0.05). The apoptotic rate of SMMC-7721 cells after treatment with SCAVB6 for 48 h was higher than that of the SMMC-7721 cells without any treatment, but the invasion ability was weakened (both P < 0.05). The expression levels of HPA mRNA and protein in SMMC-7721 cells after treatment with 1.0, 1.5 and 2.0 μg/mL SCAVB6 were lower than those in the SMMC-7721 cells without any treatment, and this inhibition effect was in a dose-dependent manner (all P < 0.05). Conclusion: SCAVB6 can inhibit the proliferation and invasion of SMMC-7721 cells. This effect may be related to the inhibition of expression of HPA.
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Objective: To investigate the effects of anti-heparanase monoclonal antibody (anti-HPA mAb) in combination with paclitaxel (PTX) on proliferation, migration and invasion of breast cancer MCF-7, MCF-7/F5 and MDA-MB-231 cells. Methods: The expression levels of HPAmRNA in MCF-7, MCF-7/Fs and MDA-MB-231 cells after treatment with anti-HPA mAb were detected by reverse transcription-PCR (RT-PCR). The proliferation of the three breast cancer cell lines after treatment with different concentrations of anti-HPA mAb or PTX alone or in combination was detected by MTT assay. The cell cycle distribution and the abilities of migration and invasion of these breast cancer cells after different treatment modalities were examined by flow cytometry (FCM), wound healing assay and Transwell assay, respectively. Results: The expression levels of HPA mRNA in MCF-7/F5 and MDA-MB-231 cells after treatment with 10 and 20 nmol/L anti-HPA mAb were lower than those of murine immunoglobulin C (IgG) treatment group (all P < 0.05). The proliferation of MCF-7, MCF-7/F5 and MDA-MB-231 cells after treatment with anti-HPA mAb or PTX alone was inhibited in a concentration-dependent manner (all P < 0.05). The proliferative inhibition rates of the three breast cancer cell lines after treatment with combination of anti-HPA mAb and PTX were higher than those of any single drug treatment groups (all P < 0.05). The ratios of G0/G1 phase of the three breast cancer cell lines after treatment with combination of anti-HPA mAb and PTX were lower than those of the control groups (three breast cancer cell lines without any treatment), and the ratios of G2/M phase were opposite (P < 0.05, P < 0.01), with a typical sub-diploid peak which appeared before C0/G1 phase. The abilities of migration and invasion of MCF-7/F5 and MDA-MB-231 cells after treatment with combination of anti-HPA mAb and PTX were lower than those of the control, murine IgG and single drug treatment groups (all P < 0.05). Conclusion: Anti-HPA mAb in combination with PTX have synergistic inhibitory effect on the abilities of proliferation, migration and invasion of breast cancer cells, and this mechanism may be associated with the arrest in G2/M phase.
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Objective To explore effect of quercetin on growth of cervical cancer,and its relationship to expression heparanase, and to investigate the possible mechanism of the inhibition effect of quercetin on cervical cancer .Methods HeLa cells were injected into nude mouse to establish the model of cervical cancer.30 nude mice with palpable tumors were randomly assigned to five groups:25,50,100 mg/kg quercetin groups,PI-88 positive group and blank control group.Transmission electron microscope was used to observe the ultrastructural changes of the vascular endothelial cells in each group.The expression of heparanase protein was detected by Western blot.Used blank control group for grey reference, relative grey value of each group was calculated for the further statistical analysis.Results 50,100 mg/kg quercetin groups and group of PI -88 made the volume of tumor tissues smaller.In 50 mg/kg quercetin group, swelling of VEC and mitochondria in tumor tissues was observed.In 100 mg/kg quercetin group, the disappearance of membraneaceous structures was found.In PI-88 positive group, the atresia of lumen, vacuole-shaped smooth endoplasmic reticulum and a dead cell were observed.50 and 100 mg/kg quercetin groups decreased the heparanase protein levels significantly(P<0.05).Furthermore, PI-88 positive group could decrease the heparanase protein levels significantly(P<0.05), compared to 50 and 100 mg/kg quercetin groups.However, the difference between the 25 mg/kg quercetin group and blank control group showed no statistical significance.Conclusion The growth of cervical cancer could be inhibited by 50, 100 mg/kg quercetin.Meanwhile, the ultrastructural changes of tumor endothelial cells could be induced by 50,100 mg/kg quercetin.These changes may relate with the decrease of heparanase protein level .
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Objective To establish a novel TRFIA for the measurement of heparanase (HPA) in serum samples,and investigate its clinical application.Methods The micro-pore plate wells were first coated with partially recombinant murine anti-human HPA monoclonal antibody.Biotin-labeled recombinant HPA protein was then used to compete with HPA in serum samples,and the prepared europium (III)-labeled streptavidin (Eu3+-SA) was used as signal readout for establishing the BSA-TRFIA assay.Using this assay,the serum HPA levels in healthy subjects (n=32) and tumor patients (n=54) were measured.The results of BSA-TRFIA were compared with those of ELISA.Two-sample t test (or t' test),and linear correlation analysis were used to analyze the data.Results The sensitivity of BSA-TRFIA for measuring HPA was 0.33 ug/L.The CV values for intra-batch and inter-batch were 5.29% and 7.54%,respectively.The average recovery rate was 105.5%.The standard curve range was 0-1 000 ug/L.The serum HPA level measured by the BSA-TRFIA method in healthy subjects was (2.03_+ 1.47) Iug/L.In tumor patients,the HPA level was significantly higher:(22.13_+7.38) ug/L (t'=19.388,P
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Objective:To explore the effect of heparanase (HPSE) on the cell adhesion and invasion ability of hepatoma carcino-ma (HC) cell. Methods:HPSE expressions in human HC cell lines (BEL-7402, HepG2, and HCCLM3) were measured by real-time re-verse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Four recombinant miRNA vectors, pcD-NATM6.2-GW/EmGFP-miR-HPSE (pcDNA-miR-HPSE), were constructed and transfected into HCCLM3 cells. Full-length cDNA of HPSE gene was cloned into pIRES2-EGFP vector and transfected into HepG2 cells. Transfection efficiency was observed with fluores-cence microscope. HPSE expressions in transfected cells were measured by real-time RT-PCR and Western blot analysis. Adher-ence ability was determined with microplate reader, and invasion and migration abilities were detected with Transwell chambers. Re-sults:Both HPSE mRNA and protein relative expression levels were higher in the three types of HC cells than those in normal hepato-cyte (P<0.05). HPSE had the highest expression level in HCCLM3 cells and the lowest expression level in HepG2 cells (P<0.05). All five recombinant vectors met the experimental requirements. The transfection efficiencies were 75%-85%. The four miRNA vectors, pcDNA-miR-HPSE, significantly decreased HPSE expression in transfected HCCLM3 cells (P<0.05), and pcDNA-miR-HPSE-1 showed the best interference effect (P<0.05). Plasmid pIRES2-EGFP-HPSE increased HPSE expression in transfected HepG2 cells (P<0.05). After pcDNA-miR-HPSE-1 was transfected, the HCCLM3 cell adherence rate and the cell invasion and migration numbers dropped by almost 50%(P<0.01). After transfection of pIRES2-EGFP-HPSE, the HepG2 cell adherence rate and the cell invasion and migration numbers increased by nearly 40%(P<0.05). Conclusion:Different HPSE vectors could regulate bi-directionally the adher-ence, invasion, and migration abilities of transfected HC cells. HPSE may be related with adherence aside from invasion of HC cell.
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Objective To observe the impact of heparanase on glomerular endothelium glycocalyx during sepsis and to investigate the prevention of glycocalyx injury.Methods C57/BL6 mice were injected with lipopolysaccharide (LPS) or tumor necrosis factor-α(TNF-o) and sacrificed one hour later.Glomerular endothelium glycocalyx traced with lanthanum was observed by transmission electron microscope(TEM).Western blotting was used to observe heparanse protein expression of renal cortex tissue.Human renal glomerular endothelial cells (HRGECs) were stimulated with TNF-α and active heparanase protien expression was detected by Western blotting.Mice were administrated with heparin sodium or heparinase Ⅲ and renal endothelium glycocalyx was observed by TEM.Urine during twenty-four hours was collected to measure urinary albumin and creatinine.The ratio of albumin to creatinine was calculated and compared among groups.Results The glomerular endothelium glycocalyx of LPS group and TNF-α group was degradated and the one of podocyte was integrated.Renal cortex tissue heparanase protein expression was significantly increased since one hour after LPS injection (P ＜ 0.01).The protein expression of activited heparanase of HRGECs which were stimulated with TNF-α was increased (P ＜ 0.05).Administration of heparin sodium which could inhibit the activity of heparanase could prevent the glycocalyx form degradation.The ratio of urine albumin to creatinine of heparin sodium group was decreased compared with LPS group (P ＜ 0.05) and the ratio of heparinase Ⅲ group was higher than control group(P ＜ 0.01) as a result of degradation of glomerular endothelium glycocalyx.Conclusions During the early stage of sepsis,TNF-α can induce glomerular endothelium heparanase to increase and active,and consequently the glycocalyx is degradated which leads to albuminuria.Inhibition of heparanase can protect glomerular endothelium glycocalyx and prevent albuminuria.
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Objective To investigate the expression of matrix metalloproteinase-2 (MMP-2)and matrix metalloproteinase-9(MMP-9) in MIA PaCa-2 cells blocked by AS-ODN cultured in hypoxia.Methods Heparanase(Hpa) expression of MIA PaCa-2 cells was blocked by AS-ODN and cultured in hypoxia.The expression of MMP-2 and MMP-9 mRNA and proteins in cell lysate was evaluated by RT-PCR and Western blot respectively,and the enzymatic activities of MMP-2 and MMP-9 in supernatants were detected by gelatinase activity assay.Results Hypoxia stimulated mRNA and protein expression of MMP-9 in cultured MIA PaCa-2 cells and elevated at 6h,12 h(P ＜0.05)and 24 h(P ＜ 0.01).When Hpa expression was inhibited by AS-ODN,the expression of MMP-9 mRNA and protein as well as the gelatinase activity in supernatant decreased dramatically at 12 h and 24 h,especially at 24h(P ＜0.01),however,no significant difference of MMP-2 expression and gelatinase activity was observed after AS-ODN transfection.(P ＞ 0.05).Conclusion In hypoxia,MMP-9 expression,either mRNA or protein in cultured MIA PaCa-2 cells,increased gradually accompanied with elevated gelatinase activities.When the heparanase expression was inhibited,the MMP-9 mRNA and protein,as well as the gelatinase B activity in supernatant,were decreased dramatically at 12h and 24h,however,no significantly differences of MMP-2 expression and gelatinase A activity were observed after the AS-ODN transfection.
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Objective To observe the alteration of glomerular anionic sites and renal heparanase (Hpa)expression in respiratory syncytial virus(RSV) nephropathy in rats,and to investigate the role of Hpa in the pathogenesis of proteinuria in RSV nephropathy.Methods Twenty-five Sprague-Dawley (SD) rats were inoculated with 6 x 106plaque forming units(PFU) RSV and were sacrificed on days 4,8,14 and 28 post-inoculation(RSV4,RSV8,RSV14 and RSV28 group).Five normal SD rats served as normal control.The proteinuria and serum parameters were measured.Glomerular anionic sites were measured by histochemical electron microscopy.The expression of Hpa in kidney was determined by immunohistochemical staining.The relationship between the expression level of Hpa and the quantity of 24-hour urine protein was studied.Results After inoculation,the proteinuria increased,especially in RSV14 group.The 24-hour urine protein of RSV14 group,RSV8 group,RSV28 group,RSV4 group and normal group were (30.9860 ± 3.3464)mg,(15.3212 ± 1.2249) mg,(13.9193-2.1409) mg,(11.5857± 1.5705) mg,(3.9780 ± 0.6224) rag,respectively.The serum albumin of RSV14 group decreased.The anionic sites of glomerular basement membrane(GBM) decreased in RSV nephropathy.The number of anionic sites per 1000 nm GBM of RSV14 group,RSV8 group,RSV28 group,RSV4 group and normal control group were 12.0000 ± 1.5811,14.0000 ± 1.0000,14.6000 ± 1.1401,16.8000 ± 0.8366 and 21.2000 ± 1.3038,respectively.Hpa in glomeruli couldn't be detected in normal rats.Glomerular Hpa expression was up-regulated in RSV nephropathy.The expression of glomerular Hpa of RSV14 group,RSV8 group,RSV28 group and RSV4 group were 0.6622 ±0.1145,0.5511 ± 0.0257,0.3524 ± 0.0296 and 0.4521 ± 0.0087,respectively.The expression level of Hpa in RSV8 group and RSV14 group was higher than that in RSV4 group and RSV28 group.There was a linear positive correlation between the expression level of glomerular Hpa and the quantity of 24-hour urine protein (r =0.783,P ＜ 0.05).Conclusions The increased expression of glomerular Hpa in RSV nephropathy of rats leads to loss of the glomerular anionic sites and damage of the electrostatic barrier of GBM,which promote the proteinuria.
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Objective To explore the clinical value of serum cathepsin L (CL),matrix metalloproteinase-9 (MMP-9) and heparanase (Hpa) detection in determining the degree of ovarian cancer invasion and metastasis.Methods Enzyme-linked inmunosorbent assay (ELISA) and electrochemiluminescencl immunoassay (ECLIA) were used to detect the serum content of MMP-9,Hpa,CL in 217 cases with untreated ovarian cancer before surgery( in FIGO Ⅰ - Ⅱ stage 83 cases,Ⅲ-Ⅳstage 134 cases),100 cases with benign ovarian tumors and 101 healthy women control.All of the patients from Guangxi Medical University Affiliated Tumor Hospital,from September 2003 to October 2009.The relationship between the clinical pathological factors of ovarian cancer and serum content of MMP-9,Hpa,CL was analyzed.On the basis of clinical pathological diagnosis as “gold standard”,the ROC curves was drawed to evaluate the clinical value of serum CL,MMP-9,Hpa combined detection in determining the degree of ovarian cancer invasion and metastasis before surgery.Results The serum content of CL,MMP-9 and Hpat in patients with ovarian cancer were (21.23 ± 8.17),( 193.95 ± 42.49),(7.68 ± 2.32) μg/L respectively,which was higher than that in patients with benign ovarian tumors [ ( 10.97 ± 3.84),( 143.66 ± 28.47),( 4.86 ± 1.37) μg/L respectively ] and normal control [ (5.59 ± 1.75),( 57.99 ± 1 1.42),( 2.77 ± 0.80) μg/L respectively ],there was difference statistically significant ( t value CL was - 13.242,- 13.498 respectively; MMP-9 was - 14.521 and - 21.290 respectively; Hpa was - 10.896 and - 18.280 respectively,P ＜ 0.001).The serum content of CL [ ( 21.59 ± 8.24) μg/L ] in patients with epithelial ovarian cancer ( EOC) was significantly higher than that [ ( 19.57 ± 7.69) μg/L ] in non-epithelial carcinoma ( F =1 1.209,P =0.048).The serum CL,MMP-9 and Hpa content in FIGO Ⅰ -Ⅱ stage patients was (19.66 ± 7.83),(182.63 ±42.30),(7.21 ±2.05) μg/L,which was lower than that (22.64 ±8.31),(202.81 ±39.74),(8.51 ± 1.92) μg/L in FIGO Ⅲ-Ⅳ stage patients ( F value was 12.452,70.565 and 195.122respectively,P value was 0.030,0.002 and 0.000 respectively).In patients with EOC,the serum CL,MMP-9 and Hpa content in eases with poorly differentiated was ( 23.04 ± 7.67),( 200.12 ± 40.82),(8.22 ± 1.92) μg/L respectively,which was also higher than that in cases with high-moderate differentiated [ ( 18.54 ± 7.30),( 173.43 ± 39.37),(7.20 ± 2.51) iμg/L respectively;F value was 24.545,60.286 and 9.077 respectively; P was 0.004,0.035 and 0.001 respectively ].The serum content of CL and MMP-9(22.96 ± 8.41),(200.44 ±43.82) μg/L respectively in patients with invasion and metastasis in the abdominal cavity was higher than that without invasion and metastasis in the abdominal cavity [ ( 19.07 ±7.36),( 181.04 ± 36.10) μg/L,F value was 12.210,18.084 ; P value was 0.030,0.010 ] ; There was statistically significant relatioship between serum levels of Hpa and patients with distant metastasis ( F =9.430,P =0.042).On base of pathological diagnosis as gold standard,ROC curve showed the sensitivity was 60.9％ (70/115),69.6％ ( 80/115) and 72.2％ ( 83/115) and specificity was 57.4％ ( 26/62),67.2％(20/62) and 68.9％ (19/62),as serum levels of CL,Hpa,MMP-9 preoperative were detected as tumor markers to determine whether there was cancer invasion and metastasis outside the pelvis.Conclusions There is related with CL,MMP-9 and Hpa levels increase and tumor occurrence and progression in ovarian cancer.The serum content of MMP-9,Hpa,CL detection would be certain clinical reference value to determine extent of invasion and metastasis of ovarian cancer before surgery.
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Objective To explore the influence on the expression of elF4E and heparanase by antisense oligodeoxyribonucleotides (ASODN) transfection in human bladder carcinoma BIU-87 cells.Methods After transfecting the 2.5,5.O,7.5 μg/ml eIF4E ASODN into BIU-87 cells,at 24 h,48 h and 72 h,a cell control group (no transfection),a blank control group (empty liposomes) and a non-sense control group (transfected with non-sense ASODN) were established.The expression of eIF4E,HPA and mRNA were detected by in situ hybridration.The expression of eIF4 and HPA protein were detected by immunocytochemistry.SPSS 13.0 statistical software was used for statistical analysis.Results The expression quantity of eIF4E protein and mRNA were lower in the experimental groups ( protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:3.55 ±0.52,3.52 ±0.51,3.22.±0.44 respectively; 5.0 μg/ml group:3.43 ±0.47,3.41 ± 0.46,3.19 ± 0.41 respectively ; 7.5 μg/ml group:2.36 ± 039,2.33 ± 0.37,2.05 ± 0.30 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:3.19 ±0.41,3.13 ±0.39,2.90 ±0.38 respectively ; 5.0 μg/ml group:3.07 ± 0.39,2.94 ± 038,2.27 ± 0.37 respectively ; 7.5 μg/ml group:2.22 ± 037,2.06 ± 0.30,1.95 ± 0.29 respectively.All data were less than those in the control groups (P ＜0.05).The expression quantity of HPA protein and mRNA were lower in experimental groups (protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:4.44 ±0.55,4.40 ±0.54,3.99 ±0.52 respectively; 5.0 μg/ml group:4.41 ±0.55,4.21 ±0.53,3.77 ±0.50 respectively; 7.5 μg/ml group:4.02 ±0.52,3.98 ±0.52,2.32 ±0.37 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:4.12 ±0.51,3.75 ± 0.50,3.63 ± 0.45 respectively ; 5.0 μg/ml group:4.00 ± 0.51,3.71 ± 0.50,3.54 ± 0.44respectively ; 7.5 μg/ml group:3.87 ± 0.52,3.61 ± 0.49,2.08 ± 0.30 respectively).All data were less than in control groups ( P ＜ 0.05 ).There was a positive correlation on expression of HPA protein and eIF4E protein by inhibitory effect of eIF-4E ASODN (protein r=9.23,mRNA r=9.59,P ＜0.01).Conclusion eIF-4E ASODN might be used to inhibit the expression of eIF-4E gene and HPA gene in bladder cancer BIU-87 cells.
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Heparanase, an endo- β- D- glucuronidase, that cleaves heparan sulfate chains in heparan sulfate proteoglycans, has recently been found in mammalian. It may play a key role in tumor progression and metastasis. Here we disscuss its molecular structure, biochemical properties, expression in malignant tumor,mechanism in accelerating tumor metastasis and application in heparanase inhibitor.