The Research Of Hpa On The Invasiveness And Metastasis Of Uterine Cervical Cancer

The uterine cervical cancer is one of the most common malignant tumors among women worldwide.The incidence of all invasive cervical cancer and of cervical squamous cell carcinoma has been decreasing but its incidence in young women has increased in developed countries,even those with widespread screening programs and histology-specific cancer registration.More and more young women want to maintain fertility after the treatment .Thus the metasis and recurrence of the tumor become the main cause of the mortality.To identify the principium of invasion and metastasis and to search effective approaches to prevent the recurrence and metastasis of the cancer remain a challenge to modern medical science.Heparanase(Hpa) degrades heparan sulfate proteoglycans(HSPGs)to facilitate tumor invasion,metastasis and angiogenesis,which are the main components of the extracellular matrix and basement membrane.It is the only one we know by now that degrades the heparin sulfate chains of HSPGs,destroies the integrity of the ECM,and also releases and activates growth factors combined with HSPGs such as bFGF,VEGF et al,and induces angiogenesis.In this way,Hpa promotes tumor invasion and metastasis.It is becoming a new target to treat cancer with the development of the study about the relationship between the Hpa and the tumor invasion and metastasis.RNA interference (RNAi) is a powerful tool for the analysis of gene function in many organism by introducing double-stranded RNA(dsRNA) into cells and leading to tha sequence specific degradation of endogenous RNAs that match the dsRNA.The secreening and identification of specific RNAi sequence is the key step of RNAi experiment.The first part of the research is on the clinicalpathological significance of Hpa protein and mRNA expression in uterine cervical cancer;The second part is the screening and identification of the target site of Hpa geng by siRNA which were synthesized in vitro system.;The third part is to investigate the change of the adhesion,invasion and metastasis ability and relative angiogenesis factors in Hela cell lines after the silencing of Hpa gene.Materials and Methods1.The expression, correlation and clinical significance of Hpa mRNA and protein in 46 patients with uterine cervical carcinoma, 10 patients with CIN and 10 patients with normal cervix were detected by histoimmunological SABC and reverse transcriptase polymerise chain reaction(RT-PCR) methods.2.Four pairs of specific targeted sequences were synthesised in vitro.Lipofectamine 2000 mediated transient transfection was conducted to transmit the siRNA into Hela cells.The differences in expression of Hpa mRNA and protein between the transfected and nontransfected cell lines were assessed by RT-PCR and Western blot method.3.Lipofectamine 2000 was used to transfect specific siRNA into Hela cells.Changes of adhesive ability of cells was detected by cell adhesion assay.Changes of invasive and chemotactic mobile capacity were measured by use of transwell-room and millicell-room. And the expression of bFGF,VEGF protein were tested by Western blot.Results1. The expression of Hpa gene mRNA and protein was significantly higher in uterine cervical cancers than that in CIN and normal cervical tissues (P<0.01) .The expression of Hpa gene mRNA and protein in late stage of cervical cancer was significantly higher than that in early stage. (P<0.01), The expression of Hpa gene mRNA and protein in the patients whose lymph nodes positive was significantly higher than those whose lymph nodes negative(P<0.05), but the expression has no relationship with the pathological stages and histological differentiation of cervical cancers. The expression of Hpa gene mRNA was correlates with their clinical stage and lymph node metastasis.2.Four pairs of specific targeted sequences were selected in specific region of Hpa gene.Transfection of Hpa siRNA was conducted with Lipofectamine 2000 in Hela cell lines.The Hpa gene expression was assessed by RT-PCR and Western Blotting assays.The result showed that siRNA1# and siRNA4# can silence the mRNA expression by 64%,67%and can silence the protein expression by 62%,61%and in a dose dependent manner. The expression of Hpa RNA and protein had no significant change with extend of transfection time.3.siRNA1# was used as specific interference group .The adhesion,invasion and chemotactic mobile capacity of Hela cells are decreased after transfected the specific Hpa siRNA into the cell lines. The expression of bFGF,VEGF protein decreased in specific interference group.Conclusions1.The higher expression of Hpa mRNA and protein maybe correlate with the invasion and matastasis of uterine cervical cancers.These findings suggest that Hpa may play a crutial role in uterine cervicai cancer invasion and could be a useful target for theraputic intervention.2.The RNAi phnomena were induced in Hela cells with the transfection of siRNAs which were synthesized in vitro.The siRNA1# and siRNA4# can silence the mRNA expression and protein expression of Hpa gene.The Hpa expression can be effectively suppressed by siRNAs.3.The specific Hpa siRNA can decrease the adhesion,invasion and chemotactic mobile capacity of Hela cells significantly after transfection. The expression of bFGF,VEGF protein were greatly decreased.Hpa gene may play a key role in the metastasis cascade.