【作者】 林颢；

【导师】 郑秀；

【作者基本信息】 福建医科大学 ， 妇产科学， 2009， 硕士

【摘要】 目的:构建S期激酶相关蛋白2(Skp2)特异性RNA干扰慢病毒表达载体,抑制子宫内膜癌HEC-1-A细胞中Skp2基因的表达。研究Skp2基因沉默后P27、CyclinD1、Caspase-3表达的变化,以及对HEC-1-A细胞周期、凋亡和增殖的影响。方法:构建U6启动子驱动、带有绿色荧光蛋白(GFP)标志的、表达针对Skp2基因的慢病毒LV-shRNA载体,转染HEC-1-A细胞,同时以转染阴性载体的HEC-1-A细胞作为对照。荧光显微镜观察GFP表达以确定转染效率,采用RT-PCR、荧光实时定量PCR在mRNA水平检测Skp2基因表达的变化,采用Western blotting在蛋白质水平检测Skp2基因表达的变化,以明确RNAi的抑制效率。并运用同样的方法分别从mRNA和/或蛋白质水平检测P27、CyclinD1、Caspase-3表达的变化,通过细胞计数、CCK-8法、流式细胞技术检测Skp2 RNA干扰后细胞增殖、凋亡和细胞周期的变化,分析Skp2基因沉默对P27、CyclinD1、Caspase-3表达水平以及细胞增殖、凋亡和细胞周期的影响。结果:1.成功构建了四对针对Skp2基因不同位点的RNAi慢病毒表达载体,经PCR和测序鉴定干扰片断的碱基序列及插入位点完全正确;2.在293T细胞中包装慢病毒,获得高滴度慢病毒上清;3.利用重组慢病毒载体将Skp2 shRNA转导入子宫内膜癌HEC-1-A细胞中,其转染效率可达到70%以上;4.构建的重组慢病毒LV-Skp2-1、LV-Skp2-2、LV-Skp2-3、LV-Skp2-4无论在mRNA水平还是在蛋白质水平均能抑制Skp2基因的表达,其中LV-Skp2-3、LV-Skp2-4的基因沉默效果较好,抑制率可以达到50%以上;5.利用RNAi技术沉默Skp2基因表达,对p27、cyclinD1的mRNA表达水平没有影响,却可以在蛋白质水平上调P27的表达,并下调CyclinD1的表达;6.抑制Skp2基因表达影响HEC-1-A细胞周期的进程,使其发生G2/M期阻滞;7.下调Skp2基因表达抑制了HEC-1-A细胞增殖,使其生长速度减缓。8.抑制Skp2基因表达对HEC-1-A细胞凋亡没有显著影响。结论:利用RNA干扰技术,可有效下调Skp2基因的表达,引起P27蛋白表达的上调和CyclinD1蛋白表达的下调,但对p27、cyclinD1的mRNA表达水平没有影响。Skp2基因的沉默影响HEC-1-A的细胞周期,发生G2/M期阻滞,进而抑制了细胞的增殖,使细胞的生长速度减慢,然而对细胞凋亡却没有太大的影响。Skp2有望成为子宫内膜癌辅助基因治疗的潜在靶点。更多还原

【Abstract】 Objective: To construct lentiviral vectors of RNA interference (RNAi) specific to S-phase kinase associated protein 2 (Skp2) and to inhibit the expression of Skp2 gene in endometrial carcinoma cell HEC-1-A, and to investigate the effects on the expression of p27, cyclinD1 and caspase-3, as well as cell cycle, apoptosis and proliferation of endometrial carcinoma cell HEC-1-A.Methods: Lentiviral vectors (LV-shRNA), containing U6 promoter and green fluorescent protein (GFP), were constructed to deliver small hairpin RNA (shRNA) targeting Skp2 into HEC-1-A cells. The control group was the HEC-1-A cells transfected with negative vector. The transfection efficiency was determined by detecting the GFP expression with the fluorescent microscopy. The expression level of Skp2 mRNA was examined by reversed transcript polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (RT-qPCR). The expression level of Skp2 protein was finally examined by Western blotting for identifing the inhibitory efficiency of RNAi. In order to analyze the effects of silencing Skp2 gene on the expression of p27,cyclinD1 and caspase-3 ,as well as cell cycle, apoptosis and proliferation, the methods mentioned above was applied to detect the changes in expression of p27,cyclinD1 and caspase-3 on mRNA and/or protein level respectively. For the same purpose, cell counting, CCK-8 method and flow cytometry were performed to examine the changes in cell cycle, apoptosis and proliferation after RNAi targeting Skp2.Results: 1. Four RNAi lentivirus expression vectors targeting to four various sites of Skp2 gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay; 2. The lentivirus were packaged in 293T cells with high titer; 3. Small hairpin RNA (shRNA) targeting Skp2 was transfected into endometrial carcinoma cells HEC-1-A by recombinants of lentiviral vectors and the transfection efficiency could reach more than 70%; 4.The expression of Skp2 gene was inhibited by lentiviral recombinants of LV-Skp2-1,LV-Skp2-2,LV-Skp2-3 and LV-Skp2-4. The inhibitory efficiency of LV-Skp2-3 and LV-Skp2-4 could reach over 50%, higher than the other two; 5. Silencing Skp2 gene by RNAi did not influence the expression of p27 and cyclinD1 mRNA; however, it could up-regulate the expression of P27 protein and down-regulate the expression of CyclinD1 protein; 6.Inhibiting the expression of Skp2 gene had the influence on the processes of HEC-1-A cell cycle thus induced G2/M arrest; 7.Down-regulating the expression of Skp2 gene inhibited proliferation of HEC-1-A cell and slowed down its growth velocity; 8. No obvious effect on the apoptosis of HEC-1-A cell was observed by inhibiting the expression of Skp2 gene.Conclusions: Effective down-regulating Skp2 expression by RNAi helped to up-regulate the expression of P27 protein and down-regulate the expression of CyclinD1 protein; however, its impact on the expression of p27 and cyclinD1 mRNA was not observed. Silencing Skp2 gene by RNAi influenced the processes of HEC-1-A cell cycle, induced G2/M arrest and further inhibited the proliferation of cells and slowed down their growth velocities with insignificant influence on apoptosis. Skp2 might be a potential adjuvant gene therapeutic target for human endometrial carcinoma.更多还原