【作者】 赵莉莉；

【导师】 姚智；

【作者基本信息】 天津医科大学 ， 免疫学， 2012， 博士

【摘要】 目的卵巢癌(Ovarian cancer,OVCA)是死亡率最高的妇科恶性肿瘤,早期诊断难、易侵袭转移、预后差是其高死亡率的重要原因。探讨卵巢癌侵袭转移的机制,寻找行之有效的治疗方法以提高卵巢癌患者生存率是卵巢癌治疗的热点和难点。Raf激酶抑制蛋白(Raf kinase inhibitory protein,RKIP)是一种高度保守、广泛表达的蛋白,参与细胞生长、增殖、分化、凋亡等重要的生理功能,并被证实具有肿瘤转移抑制作用。该作用最初被发现是在前列腺癌研究中,其后陆续在黑色素瘤、胃癌、结直肠癌、非小细胞肺癌、乳腺癌、肝癌等恶性肿瘤中发现RKIP表达减少或缺失,并具有肿瘤转移抑制作用。本实验室已证实RKIP是卵巢癌的转移抑制基因,但对RKIP抑制卵巢癌转移的机制并未阐明。本课题将深入研究RKIP对Raf-1/MEK/ERK信号通路的调控,从RKIP影响卵巢癌细胞粘附能力及侵袭能力的分子机制方面着手探索RKIP抑制卵巢癌转移的可能作用机制,为卵巢癌治疗提供新的靶点和方法。方法1.探讨RKIP对Raf-MEK-ERK信号转导通路的调节作用。(1)应用脂质体转染法将空载体pcDNA3.1(+)、pcDNA3.1(-)及正义表达RKIP的重组质粒pcDNA3.1(+)-ssRKIP和反义表达RKIP的重组质粒pcDNA3.1(-)-asRKIP 转染 SKOV3 细胞,G418 筛选阳性克隆,Western Blot鉴定RKIP蛋白水平,选取高表达RKIP和低表达RKIP的稳定细胞株。根据转染的质粒不同将细胞分为5组:未转染任何质粒的空白对照组,pcDNA3.1(+)组、pcDNA3.1(-)组,SsRKIP 转染组以及 AsRKIP 转染组。(2)收集各组细胞,提取细胞总蛋白,采用Western　Blot技术,检测各组细胞中磷酸化 MEK(phospho-MEK,p-MEK)、磷酸化 ERK(phospho-ERK,p-ERK)蛋白的表达水平,漂洗NC膜后再分别检测总MEK(total MEK)和总ERK(total ERK)蛋白表达水平。2.研究RKIP对SKOV3细胞粘附能力的影响。(1)利用体外细胞粘附实验研究RKIP表达对SKOV3细胞与Matrigel胶体外粘附能力的影响。(2)应用Real-time PCR、Western Blot方法分别观察上调或抑制卵巢癌细胞中RKIP蛋白表达后对细胞表面粘附分子CD44、ICAM-1及E-Cadherin mRNA、蛋白表达的影响。3.研究RKIP对SKOV3细胞侵袭能力的影响。(1)应用细胞划痕试验检测上调或抑制RKIP表达对SKOV3细胞迁移能力的影响。(2)应用Transwell小室体外侵袭实验检测上调或抑制RKIP表达对SKOV3细胞侵袭能力的影响。(3)应用Real-time PCR、Western Blot方法检测上调或抑制RKIP表达后对卵巢癌细胞MMP-2和MMP-9mRNA和蛋白表达的影响。(4)应用明胶酶谱分析法测定上调或抑制RKIP表达对卵巢癌细胞MMP-2和MMP-9蛋白酶活性的影响。结果1.探讨RKIP对Raf-MEK-ERK信号转导通路的调节作用。(1)筛选出正义表达RKIP和反义表达RKIP的稳定细胞株,其中SsRKIP转染组的SsRKIP#5、SsRKIP#7、SsRKIP#8 RKIP蛋白表达水平分别增高为空白对照组的 2.60 倍、2.05 倍和 1.66 倍,AsRKIP 转染组的 AsRKIP#6、AsRKIP#8、AsRKIP#10 RKIP蛋白表达水平分别降低为空白对照组的32%、35%和5%。选取空白对照组,空载体pcDNA3.1(+)组和pcDNA3.1(-)组、SsRKIP转染组的三支稳定细胞株、AsRKIP转染组的三支稳定细胞株进行后续试验。(2)SsRKIP转染组细胞p-MEK、p-ERK表达低于空白对照组和/或空载体pcDNA3.1(+)组,AsRKIP转染组SKOV3细胞p-MEK、p-ERK表达高于空白对照组和/或空载体pcDNA3.1(-)组,但总MEK和总ERK在各组细胞中的表达无明显差异。2.探讨RKIP对Raf-MEK-ERK信号转导通路的调节作用。(1)RKIP可降低与Matrigel胶粘附的SKOV3细胞数,即RKIP可降低SKOV3细胞的体外粘附能力。(2)与空白对照组和/或空载体组相比较,SsRKIP转染组细胞CD44、ICAM-1 mRNA水平降低,E-Cadherin mRNA水平升高,AsRKIP转染组细胞CD44、ICAM-1mRNA和蛋白表达增加,E-Cadherin mRNA和蛋白表达下降,差异均有统计学意义(P<0.05)。(3)与mRNA结果相一致,与空白对照组和/或空载体组相比较,SsRKIP转染组细胞CD44、ICAM-1蛋白表达降低,E-Cadherin蛋白表达升高,而AsRKIP转染组细胞CD44、ICAM-1蛋白表达增加,E-Cadherin蛋白表达下降。3.研究RKIP对SKOV3细胞侵袭能力的影响。(1)细胞划痕试验显示SsRKIP转染组细胞迁移率降低,反之,AsRKIP转染组细胞迁移率增加,即RKIP可降低细胞的迁移能力。(2)SsRKIP转染组细胞体外侵袭能力低于空白对照组和/或空载体pcDNA3.1(+)组,AsRKIP转染组高于空白对照组/或空载体pcDNA3.1(-)组,差异有统计学意义(p<0.05)。(3)与空白对照组和/或空载体相比较,SsRKIP转染组细胞MMP-2/MMP-9 mRNA表达降低,AsRKIP转染组细胞MMP-2/MMP-9mRNA表达升高,差异均有统计学意义(P<0.05)(4)与空白对照组和/或空载体组相比,SsRKIP转染组细胞MMP-2/MMP-9蛋白表达、蛋白酶活性均降低,AsRKIP转染组细胞则均升高。结论体外试验证实RKIP既可抑制SKOV3细胞粘附能力也可抑制SKOV3细胞迁移能力及侵袭能力。其作用机制可能是通过抑制CD44、ICAM-1表达来降低细胞的异质性粘附能力,通过增加E-Cadherin表达来增强卵巢癌细胞间的同质性粘附能力,通过抑制MMP-2/MMP-9的表达及蛋白酶活性来抑制SKOV3细胞对细胞外基质的降解,从而抑制卵巢癌细胞自原发灶脱落及向远处转移。而RKIP发挥其功能与其对信号转导通路Raf-1/MEK/ERK的作用有关,RKIP可抑制Raf-1诱导的MEK、ERK活化,进而抑制了Raf-1/MEK/ERK信号转导通路。更多还原

【Abstract】 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.更多还原