Molecular Mechanisms Of Raf Kinase Inhibitory Protein In Modulating The Adhesion And Invasion Of Ovarian Cancer Cells

ObjectiveOvarian cancer(OVCA)is the most lethal forms of all common gynaecological malignancies and the fifth leading cause of cancer-related death among women in the Western Wold.The poor prognosis results from late diagnosis and high metastasis.It is very important to understand the mechanism of invasion and find effective approaches to prevent the metastasis.Raf Kinase Inhibitory Protein(RKIP)has emerged as a significant molecule in suppressing cancer metastasis.In humans,a reduction or loss in RKIP expression was significantly observed in metastatic lesions from prostate,melanoma,gastric cancer,colorectal cancer,non-small cell lung cancer,breast cancer,liver cancer.The function of RKIP to suppress the metaststic spread of ovarian cancer has been found in our laborary.But the little is known about the regulatory mechanisms of RKIP in ovarian caner.To investigate the possible mechanisms by which RKIP inhibits the metastasis of ovarian cancer,we observed the effect of RKIP on metastatic ability of adhesion,migration and invasion in SKOV3 cells(one of the OVCA cells)in vitro.Methods1.Expore the role of RKIP expression in the modulation of Raf-1/MEK/ERK signal transduction pathway in SKOV3 cells.(1)SKOV3 cells were transfected with recombinant plasmids pcDNA3.1(+)-ssRKIP or pcDNA3.1(-)-asRKIP which expressed sense(Ss)or antisense(As)RKIP cDNA or empty vector pcDNA3.1(+)/pcDNA3.1(-)by liprfectamine.The positive cell clones were selected by G418,and analyzed by Western Blot.Then the cells were divided into five groups:parent group that is the SKOV3 cells which were not transfected with any vector,pcDNA3.1(+)vector-transfected group,pcDNA3.1(+)vector-transfected group,SsRKIP vector-transfected group,and AsRKIP vector-transfected group.(2)The phosphorylation status of endogenous MEK or ERK was examined in SKOV3 cells and their stable transfectants by Western Blot analysis.Those membranes were subsequently stripped and then were used to detect both the phosphorylated and unphosphorylated forms of MEK and ERK.2.Observe the molecular mechanisms through which RKIP affects the adhesion ability of SKOV3 cells.(1)The effects of RKIP expression on SKOV3 cells adhesion to matrigel were observed by MTT method.(2)The effects of RKIP expression on the mRNA level of CD44,ICAM-1 and E-Cadherin were observed in SKOV3 cells by Real-time PCR.(3)The protein expressions of CD44,ICAM-1 and E-Cadherin were observed in SKOV3 cells and their transfectants by Western Blot.3.Examine the effects of RKIP expression on the migration and invasion of SKOV3 cells(1)The relationship between the RKIP expression and the ability of cell migration was observed by wound healing assay in SKOV3 cells and their transfectants.(2)The cell invasion abilities of SKOV3 cells and their transfectants was measured using an in vitro invasion assay.(3)The effects of RKIP expression on the mRNA level of MMP-2 and MMP-9 was examined in SKOV3 cells by Real-time PCR.(4)The protein expressions of MMP-2 and MMP-9 were observed in SKOV3 cells and their transfectants by Western Blot.(5)The effects of RKIP expression on MMP-2 and MMP-9 proteinase activities of SKOV3 cells by zymography.Results1.Expore the role of RKIP expression in the modulation of Raf-1/MEK/ERK signal transduction pathway in SKOV3 cells.(1)SKOV3 clones stably expressing SsRKIP,AsRKIP,and their respective empty vector were obtained.The SsRKIP vector-transfected SKOV3 cells demonstrated increased RKIP expression compared with the patent group or empty vector transfected goup.In contrast,the expression of RKIP was decreased in AsRKIP vector-transfected SKOV3 cells.(2)The SsRKIP vector-transfected group had less phosphorylated MEK and ERK than did the parent group and/or pcDNA3.1(+)vector-transfected group.The AsRKIP vector-transfected group had more phosphorylated MEK and ERK than did the parent group and/or pcDNA3.1(-)vector-transfected group.But the total MEK or ERK was similar in each group.2.Observe the molecular mechanisms through which RKIP affects the adhesion ability of SKOV3 cells.(1)The ability of cell adhesion in the SsRKIP vector-transfected group was decreased compared with the parent group and/or pcDNA3.1(+)vector-transfected group,whereas that in the AsRKIP vector-transfected group was increased compared with the parent group and/or pcDNA3.1(+)vector-transfected group.(2)Compared with the parent group and/or respective empty vector groups,the mRNA level and protein expression of adhesion molecules CD44,ICAM-I and E-Cadherin were repressed in SsRKIP vector-transfected group.Meanwhile,the mRNA level and protein expression of adhesion molecules were increased in AsRKIP vector-transfected group.3.Examine the effects of RKIP expression on the migration and invasion of SKOV3 cells(1)The effect of RKIP on the migration of SKOV3 cells was analyzed by would healing assay and the results revealed that SsRKIP vector-transfected group had a significantly higher migration velocity than the parent group.Conversely.AsRKIP vector-transfected group had a lower migration velocity.(2)Overexpression of RKIP led to decreased invasiveness in different Ss-RKIP SKOV-3 clones compared with parent group and/or pcDNA3.1(+)vector-transfected group.Conversely,down-regulated RKIP expression caused the increase in in vitro invasive ability in different As-RKIP SKOV-3 clones.(3)Compared with the parent group and/or respective empty vector groups.SsRKIP vector-transfected group exhibited significant down-regulation of MMP-2 and MMP-9 at the mRNA level and protein expression.Meanwhile,the mRNA level and protein expression of MMP-2 and MMP-9 were increased in AsRKIP vector-transfected group.(4)A strong decrease of matrix metalloprotease activity was observed in SsRKIP vector-transfected group compared with parent group and/or pcDNA3.1(+)vector-transfected group.On the contrary,the matrix metalloprotease activity was increased in AsRKIP vector-transfected group.ConclusionThese results suggest that RKIP could be inversely associated with the abilities of cell adhesion,migration an invasion of SKOV3 cells in vitro.The mechanism may be as follows:RKIP could inhibit the heterogeneity adhesion through repressing the expression of CD44 and ICAM-1,and enhance the homotypic adhesion through increasing the expression of E-Cadherin.In addition,RKIP might play a negative regulation role in the metastasis of SKOV3 cells through decreasing the expression and proteolytic activities of MMP-2 and MMP-9.Furthermore,the invasive and metastatic capability of SKOV3 cells suppressed by RKIP expression might be associated with the activation of MEK and ERK,which can be ihibited by RKIP expression.