Investigation The Mechanism Of WWOX In Invasiveness And Metastasis Of Ovarian Cancer

Background and Objectives:Ovarian cancer is one of the most severe female genital tumors which has tendency of extensive metastasis and spreading within pelvic and peritoneal cavity.At the moment of diagnosis,about 70%of patients displays at advanced stage with peritoneal dissemination metastasis.The mortality is highest in gynecological malignancies.Numerous investigations have demonstrated that invasion and metastasis is important factor affecting on prognosis. Metastasis is a complicate process involving in multiple factors and multiple steps,controlled by many genes.The attachment of carcinoma cells is initial factor in the process of the invasiveness and metastasis.Adesion is a key step in the initial process of peritoneal dissemination.Carcinoma cells attach initially the basement membrane and extracellular matrix(ECM)components via cell surface receptors,then proteases hydrolyze the basement membrane and ECM,invade straightly through basement layer and stroma.Integrins,an important family of adhesion molecule in cell surface receptors,mediate cell-extracellular matrix adhesion.Integrins consist of two subunits,αandβ.The most basic adhesive function of integrin play a critical role in metastasis.WWOX is recently cloned tumor suppressor gene that spans the common chromosomal fragile site FRA16D at chromosome area 16q23.3-24.1.This region is frequent target for loss of heterozygosity and homozygous deletions.WWOX is altered in several cancer types, including breast,prostate,ovarian,lung,pancreatic and gastric carcinoma.DNA instability at common fragile sites is associated with cancer.WWOX is Similar to FHIT which located at common chromosomal fragile site FRA3B,so WWOX is regarded as another new tumor suppressor gene following FHIT gene.WWOX was transfected into the ovarian carcinoma cells in order to explore its role in tumorigenesis and investigate the function of WWOX in invasiveness and metastasis of ovarian cancer. WWOX is frequently disrupted in ovarian cancer.The adhesive propensity of ovarian cancer to abdominal cavity may related to ECM.Up-regulation of ECM is associated with the loss or no function of WWOX expression.WWOX was transfected into the ovarian carcinoma cells in order to explore the interaction of WWOX protein with ECM,investigate the interaction of cells with extracellular matrix(ECM),understand the role of WWOX in the invasiveness and metastasis.Methods:1.Reverse transcriptase polymerase chain reaction(RT-PCR)was used to detect the expression of WWOX in Hct116/4 coildtype,Hct116 p53 null,Hct116 p21,Hct116 Bax null cell lines.PCR products were excised from the agarose gel and extracted using the QIAquick Gel Extraction Kit.They were sequenced after gel-purification.Sequencing results were compared to the published sequence of AF21943 in GeneBank to understand the effect of WWOX alteration on tumorigenesis.2.RT-PCR was used to detect the expression of WWOX gene,western blot also detected the expression of the WWOX protein in WWOX transfected PEO1 cells(H cells)and empty-vector transfected control cells(F cells).Western blot detected change of integrins in H cells and F cells.3.Constructed plasmid carrying WWOX or empty-vector were extracted and purified using QIAfilter Plasmid maxi kit.After Sterilization,those Constructed plasmid transiently transfected into PEO1 cells respectively.Western Blot was detected the expression of the WWOX protein and change of the integrins.4.Matrix attachment assay was used to assess the adhesion of the transiently and stable transfection in PEO1 cells via culturing the cells on the pre-coated fibronctin wells.5.Alpha or beta integrin-mediated cell adhesion assay were designed to identify cell surface integrins in PEO1 clone cells.Adhesion assay following integrin blocking were designed to further determine integrins in PEO1 clone cells according to the alpha or beta integrin that were selected.FACS analysis was determined the level of expression of various integrins on the cell membrane.Mesothelial assay was tested the adhesion of the PEO1 clone cells to mesothelial layer. 6.RNA interference(RNAi)was used to further confirmed the function of WWOX in metastasis of ovarian cancer.The A2780 cells were transfected with 50nM of the smart pool of siRNA WWOX by using Lipofectamine.After transfection 96h,Matrix attachment assay was detected the adhesion to fibronectin.Western Blot also detected the expression of WWOX silencing.7.Caspase-Glo^TM3/7 assay was used to detect the effect of WWOX knockdown on cisplatin induced apoptosis.Results:1.Sequencing of the PCR products was compared with AF211943 sequence in GeneBank. The results showed the first primer pair(9F1 and 9R1)that could amplify both transcripts, gave a product showing heterozygosity at T1497G,confirming that both alleles of WWOX are expressed.The second primer pair(8F1 and 9R2)that could amplify only the full-length transcript,gave a product with only guanine at position T1497G.The third primer pair(5F3 and 9R3)produced two products of 929bp and 389bp.Sequencing,after gel-purification,showed that only thymidine was present at position T1497G in the 389bp product that would be produced from the WWOX aberrant transcript.2.RT-PCR showed that expression of mRNA WWOX in exon9 was in the H cells and Fcells.Western Blot showed that expression of WWOX was in the H cells,but not in the F cells.The expression of theα2 orβ1 integrin in F cells were higher than that in H cells(p＜0.05).3.Matrix attachment assay showed that adhesion of F cells or parental cells were more increased than that of H cells after cells were seeded on the pre-coated fibronectin, incubated for 2h(p＜0.01).There was no significantly association in the culture for 30minutes,1h,24h(p＞0.05).4.Western Blot showed that expression of WWOX in transiently cells appeared after 24h of transfection.Expression of WWOX was highest for 72h,and then decreased gradually, almost disappeared for146h.The expression of theβ1 integrin decreased gradually with the increasing expression of WWOX,but expression of theα2 integrin was on the contrary,it increased as the expression of WWOX increased. 5.Alpha integrin-mediated cell adhesion assay showed that expression of theα2 orα3 integrin in vector transfected control lines(F cells)were markedly highter than that in WWOX-expressing PEO 1 transfectants(H cells)(p＜0.05=.6.Beta integrin -mediated cell adhesion assay showed that expression of theβ1 integrin in F cells were significantly highter than that in H cells(p＜0.05=.7.Adhesion assay following integrin blocking also showed that blockage ofα2 orα3 integrin,the number of cells decreased in the culture on pre-coated fibronctin wells, compared to the IgG control group.Blockage ofα3 integrin,the number of F ceils significantly decreased than that of H cells(p＜0.05).Biockage ofα2 integrin,there was no statistic between F cells and H cells(p＞0.05).8.Adhesion assay following integrin blocking also showed that blockage ofβ1orβ2 integrin,the number of cells decreased in the culture on pre-coated fibronctin wells, compared to the IgG control group.Blockage ofβ1 integrin,the number of F cells significantly decreased than that of H cells(p＜0.05).Blockage ofβ2 integrin,there was no statistic between F cells and H cells(p＞0.05).9.FACS assay showed theα3 expression of cell surface in F cells were higher than that of H cells(p＜0.05),but there was not any difference inα2 expression between F cells and H cells(p＞0.05).10.Matrix attachment assay showed the adhesion to fibronectin after knockdown WWOX significantly significantly increased than that in the control cells(mock,non targeting, untreated group)(p＜0.01).Western Blot also showed that WWOX protein expression was markedly decreased after silencing WWOX(p＜0.01).In cells with mock and untreated cells,no decrease in total protein expression was detected.11.Caspase-Glo^TM3/7 assay showed that cells with WWOX knockdown,compared to control group(mock and untreated cells),no association was evident on cisplatin induced apoptosis(p＞0.05).Conclusions:1.HCT116 expresses two WWOX transcripts,full length WWOX mRNA,and an abnormal transcript that lacks exons 6-8.The discovery of the heterozygous deletion that removes exons 6 to 8 suggested that the two transcripts were the products of the two alleles,each transcript originates from a distinct allele in HCT116.2.H cells is the cells that WWOX gene can be integrated to the genome of PEO 1 cells and generated stably.F cells is the cells that empty vector can be integrated to the genome of PEO 1 cells and stable generation.WWOX can down-regulate the expression of theα2,α3 andβ1 integrins,decrease the cell adhesion.WWOX can also decrease the adhesion to fibronectin,suggesting that WWOX can suppress the invasiveness and metastasis of carcinoma cells.3.Transient transfection assay further confirmed that WWOX can down-regulate the expression of theβ1 integrins,decrease the cell adhesion to fibronectin.4.Knockdown WWOX led to increase the cell adhesion to fibronectin.This further demonstrate that WWOX can down-regulate adhesion of carcinoma cells,and retard the invasiveness and metastasis.5.Silencing WWOX does not influence on cisplatin- induced apoptosis.6.WWOX is another new tumor suppressor gene.