Study Of LPA₂ Receptor On LPA-Induced UPA Secretion And Cell Invasion In Ovarian Cancer Cells In Vitro

Background:Ovarian cancer accounts for more deaths than all other gynecologic malignancies combined.Because of the obscure early symptoms and the absence of effective measures of universal inspection and early diagnosis of ovarian cancer,more than 2/3 of the patients with ovarian cancer have reached an advanced stage when they were diagnosed,with little hope to get good curative effect,and the 5-year survival is only 20%-30%,while the survival rate of patients diagnosed in early stage could reach up to 90%.Although three therapeutic advances of malignant ovarian carcinoma in the late twenty years,which are comprehensive stage laparotomy, cytoreductive surgery and omentectomy,and paclitaxel-cisplatin combined chemotherapy,cannot improve the patients' survival rate in the long run.The invasion and metastasis of ovarian cancer are the most important influencing factors to success or failure of treatment.The metastasis of tumors is a complicated process including multiple steps and factors.So it has an important significance to investigate the metastasis and invasive mechanism of epithelial ovarian cancer,and can provide new methods and potential targets for molecule treatment.LPA is a small molecular lipid functionally related with growth factors.Its signaling pathways play many biological functions mainly through G protein-coupled receptor.LPA induces proliferative and/or morphological effects.Recent years,LPA and its pathways were found to play important roles in the appearance,development, invasion and transfer of malignant tumors,such as thyroid cancer,pancreatic cancer, gastric cancer,colorectal cancer,ovarian caner and so on.To date,five LPA receptors have been found,namely LPA1/EDG-2,LPA2/EDG-4,LPA3/EDG-7, LPA4/GPR23/p2y9,and LPA5/PPARγ.LPA stimulates the proliferation,adhesion, invasion and metastasis,and the production of various invasion-associated factors through these receptors.In vitro studies have shown that LPA₁ is highly expressed in the normal and immortalized ovarian cells,while LPA₂/EDG4 and LPA₃/EDG7 mRNA are highly expressed in ovarian tissues and tumor cell lines.Thus LPA receptor is hopeful to become a new target for treatment of ovarian carcinoma.Urokinase plasminogen activator(uPA)is one of the most important enzymes to degrade extracellular matrix(ECM).The function it performs in the invasion and metastasis of tumor cells is also testified,uPA has been linked to the malignant transformation in ovarian,breast and colon cells.In ovarian cancer,cellular uPA levels correlate inversely with prognosis.In comparison with other cancers,uPA is highly expressed in ovarian cancer cell lines and ascites.uPA contributes to metastasis and migration because it catalyzes the conversion of plasminogen to plasmin,thus leading to degradation of the basement membrane.It also stimulates cellular migration and proliferation pltentially by binding to its specific cell surface receptor(uPAR).Recent research shows that LPA can induce uPA but not uPAR the secretion of uPA not uPAR and accelerate the uPA-induced invasion of tumor cells.Therefore,research on the relationship between LPA and uPA will help us understand the function of LPA in the invasion and metastasis of ovarian cancer.As a totally new way in biological field RNA interference technology has very huge potential and is getting more and more importance from the investigators.To choose the target gene which has therapeutic action on tumor is the key of the RNA interference.The studies on LPA-induced metastasis of tumor cells show that LPA₂ receptor plays an important part role in the secretion of invasion-associated cell factors in response to LPA,although other LPA receptors could also mediate the response.So we choose LPA₂ receptor to be the target gene to RNA interference.Based on the above report,this research will firstly observe the role of LPA on the production of uPA in human ovarian cancer cell line SKOV-3.Secondly,LPA₂ siRNA was transfected into SKOV-3 by using Lipofectamine^TM2000.The transfection effect was identified through detecting LPA₂ mRNA level by semiquantitative RT-PCR and protein level by Western blot,respectively. Furthermore,the role of inhibiting LPA₂ pathway in LPA-induced uPA regulation was observed,and the function of LPA2 in LPA-induced invasion and metastasis of ovarian cancer would be discussed.Partâ… :Effect of lysophosphatidic acid(LPA)on urokinase plasminogen activator(uPA)secretion of ovarian cancer SKOV-3 cellsObjective:To investigate the effect of LPA on the uPA secretion and cell invasion ability of ovarian cancer SKOV-3 cells.Methods:Human ovarian cancer cell lines were cultured in various concentrations of LPA(0,2,20,40,80μmol/L)for 24 hours and cultured in 80μmol/L LPA for different time-points up to 24 hours(0,3,8,12,24h).Then cell culture supernatant was collected,and LPA-induced uPA expression was analyzed using uPA ELISA.After LPA stimulation with the concentrations of 0 or 80μmol/L, the invasiveness of SKOV-3 cells was analyzed by using Matrigel Transwell Assay.Results:1.After 24h of incubation with various concentrations of LPA,2μmol/L of LPA can cause the increase of uPA secretion in comparison with the control group(P＞0.05).While after adding LPA with concentration of 20,40,80μmol/L,the uPA secretion increased obviously(P＜0.001).2.After incubation with 80μmol/L LPA in different time-points up to 24 hours, the secretion of uPA increased obviously in comparison with the control group after 8 hours(P＜0.01).3.The number of SKOV-3 cells that penetrated the Matrigel after 80μmol/L LPA stimulation increased obviously in comparison with control,group(219.4±23.6 vs.67±10.9)(P＜0.001).Conclusions:1.The low concentration of LPA could induce uPA secretion.With the increase of LPA concentrations,the production of uPA significantly increased,there was obvious dose-dependent manner between LPA and uPA.2.LPA treatment at 80μmol/L induced considerably uPA secretion in time-dependent manner.3.LPA could significantly increase the invasion ability of SKOV-3 cells in vitro.Partâ…¡:Effect of inhibition of LPA₂ by siRNA on LPA-induced uPA production and cell invasion of ovarian cancer SKOV-3 cellsObjective:The purpose of this study is to investigat the inhibition effect of RNA interference(RNAi)on the expression of LPA₂ in ovarian cancer SKOV-3 cells,and to observe its impact on the output of LPA-induced uPA and on cell invasion. Meanwhile,we will explore the applying prospects of RNAi technique on anti-tumor genetic treatment and the possibility that knock-down of LPA₂ gene as a treatment target of ovarian cancer.Methods:1.Ovarian cancer cell strain SKOV-3 was resuscitated and cultivated until it came to logarithmic growing period,and then they were divided into three groups, which were normal control group,negative control group and siRNA group.2.The siRNA of different concentrations of grads were transfected into SKOV-3 cells in order to observe the transfection effects,and then the optimal transfection concentration was selected.3.The specific siRNA and negative control siRNA were transfected into siRNA group and negative control group respectively.The normal control group was not transfected by any siRNA,only transfection liquid.4.The expression of LPA2 mRNA and protein were detected by using the semi-quantitative RT-PCR and Western blot after 36 hours.Meanwhile,the production of LPA-induced uPA was measured by ELISA analyse before and after the interference.The impacts of LPA on the ex-vivo invasion ability of ovarian cancer using Matrigel invasion assay.Results:1.The results of semi-quantitative RT-PCR and Western blot indicated that after transfection of LPA₂-specifical siRNA,the expression of LPA2 mRNA and protein in SKOV-3 cells were obviously reduced.2.ELISA results indicated that the production of uPA induced by LPA of 80μmol/L after interference decreased,which of siRNA group(uPA OD 450nm: 0.344±0.039)decreases obviously in comparsion with that of negative control group (uPA OD 450nm:0.746±0.031)(P＜0.001).3.The experiment of Matrigel invasion membrane indicated that the number (36.2±3.3)of cells that penetrated through Marigel chamber in siRNA group after the stimulation of 80μmol/L LPA were lower than that of negative control group (178±17.2)(P＜0.001).4.The experiment of Transwell Chemotaxis chamber indicated that the number (57±7.6)of cells that entered the lower well in siRNA group after the LPA stimulation decreased obviously than that of negative control group(220.4±25.5)(P＜0.001).Conclusions:1.The specific siRNA aiming at LPA2 gene can be successfully transfected into SKOV-3 cells and then inhibites and degrades congenetic mRNA.It can also block the expression of LPA2 mRNA and protein effectively,and can be used as a new tool of blocking LPA2 gene expression and evaluating its biological function. 2.The inhibition of LPA₂ gene by using RNAi can reduce the LPA-induced uPA secretion and the ability cell invasion and migration in SKOV-3 cells,which indicates that LPA2 is probable to work as target gene in the therapy of ovarian cancer.