The Study On Expression Of Heparanase In Epithelial Ovarian Carcinoma And Mechanism Of Tumor Metastasis

As one of the most malignant gynecologic tumors,the early symptoms of ovarian carcinoma are very secret and obscure,most of which has metastasized when diagnosed clearly.Adhesion and movement of the tumor cells,the degradation of extracellularmatrix and basementmembrane,also neovascularization are essential requisition during the transfer process.Only when the tumor cells break through this barrier can they invade and transfer into adjacent tissues or further.Heparanase(HPA)is an endoglucuronidase recently cloned that specifically degrades heparan sulfate proteoglycan(HSPG),which is a glycosaminoglycan(GAG)chain that occurs in HS proteoglycans belonging to the ECM and BM,with the potential to release cytokines and growth factors such as bFGF,VEGF and kinds of enzymes from the ECM reservoir,then accelerate the tumor invasion and angiopoiesis, eventually promoting the tumors' metastasis.As the only enzyme having the potential to degrade HS,HPA has becoming an attracting new target to anti-tumor therapy.Hypoxia is a considerable microenvironment of ovarian carcinoma, and is one of the necessary environmental conditions during the process of tumor growth.The studies have already demonstrated that hypoxia was linked to metastatic invasion through multiply mechanism, and a independent agent leading to poor prognosis of most malignant tumors.As previously discussed,hypoxia could activate nuclear transcription factor HIF-1(hypoxia inducible factor-1),the heterodimer composed of a constitutively expressed HIF-1 beta subunit and an oxygen-sensitive HIF-1alpha subunit,which was DNA binding protein that the EPO enhanser could band specially during hypoxia, and then regulate all gene transcription the hypoxia induced.The activity of HIF-1 is regulated by the abundance of alpha subunit only.Nowadays,little is known about the relationship between the HPA expression and clinical and pathologic characteristics in human epithelial ovarian carcinoma,how the expression and function of heparanase is regulated under different physiological and pathological conditions.Because of the special PH activity about HPA and local PH area hypoxia microenvironment induced in tumor,it is significant to consider whether hypoxia influence the function of HPA. The study abroad indicated hypoxia strengthened the potential of tumor cells to invade ECM,however,there is no report about the relationship between hypoxia,HPA expression and invasiveness of ovarian tumor cells.In addition,HIF-1 is nuclear transcription factor hypoxia activated,and almost regulates transcription of all the genes hypoxia induced,so it is presumed that whether hypoxia influence function of HPA through HIF-1,furthermore to strengthen the potential of ovarian carcinoma to invade.To investigate the influence mechanism of HPA on genesis,development and metastasis of human epithelial ovarian carcinoma in hypoxia situation,can help us search a new way to therapy the carcinoma by disturbing the effect of HPA.This study includes two partsPart One The Expression And Clinical Significance Of HPA In Human Epithelial Ovarian CarcinomaObjective:To investigate the relationship between expression of HPA and clinical and pathologic characteristics in human epithelial ovarian carcinoma.Methods:Western-blot was used to detect the protein expression of HPA in 65 EOC tissues and 28 benign ovarian tumors,the relationship between expression of HPA and clinical and pathologic characteristics was analyzed by x² analysesResults:1,The positive rate of HPA in 65 EOC was 70.8%,17.9%in benign ovarian tumors,the difference was significant(P＜0.01).2,The expression of HPA had significant correlation with the clinical stages and metastatic lymph node lesions(P＜0.05).3 There was no difference with the age of patients,pathologic grades and histologic types of tumors(P＞0.05).Conclusion:1,The expression lever of HPA is higher in ovarian carcinoma than in benign tumors.2,The expression of HPA has a close relationship with clinical stages and lymph node metastasis of EOC, but has not one with the age of patients pathologic grades and histologic types of tumors.3,Detecting the expression of HPA may play a important role in predicting biological behaviour of EOC.Part Two The Influence Of Hypoxia On Expression Of HPA And Related MechanismObjective:To evaluate the effects of hypoxia on the expression of HPA and the invasiveness of SKOV3 ovarian carcinoma cell line,and further to explore the relationship between HIF-1αand HPA.Methods:SKOV3 cell lines were incubated in vitro at either normoxia (37℃,5%CO₂)or hypoxia(37℃,5%CO₂,1%O₂)condition for 12h,24h and 36h.Western-blot was used to detect the protein expression of the HPA and HIF-1α,and mRNA expression was determined by RT-PCR. Cell invasiveness was measured by matrigel invasion assay.Expression of the HPA and HIF-1αwere detected after rapamycin was used.Results:Western-blot analysis revealed the protein and mRNA expression of HPA kept the increasing tendency,which was increased markly(P＜0.05)during 12-36h period.Matrigel invasion assay detected the level of cell invasion was significantly increased for 12h,24h and 36h groups(P＜0.01),meanwhile,there was a positive correlation between the expression of HPA and the invasiveness of SKOV3 ceils (r=0.9863,P＜0.01).And compared with the unchanged mRNA expression of HIF-1α,the protein expression of HIF-1αwas significantly increased (P＜0.05)under hypoxia for 12h,and remained high level for 24h,36h (P＜0.05),rapamycin couldn't chang the mRNA tendency,however,it inhibited the up-regulation of HIF-1αprotein expression,and the peak amplitude of HPA expression also was decreased abundantly after utilizing rapamycin.Conclusion:1,hypoxia promotes the expression of HPA and the invasiveness of tumor cells.2,Invasiveness of SKOV3 cells in hypoxia condition correlates with HPA level.3,The upregulated expression of HPA in hypoxia was dependent the HIF-1αpathway.The expression lever of HPA is higher in ovarian carcinoma than that in benign tumors,and has a close relationship with clinical stages and lymph node metastasis of EOC.Detecting the expression of HPA may play a important role in predicting clinical progression of EOC.The upregulated expression of HPA in hypoxia was dependent the HIF-1αpathway,and correlates with invasiveness of tumor cells.