【作者】 王鹏；

【导师】 赵洪洋；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 目的胶质瘤通常伴有p53的突变。Wip1基因抑制并负反馈调节p53功能而表现出癌基因的特性。本研究拟探讨Wip1基因在脑胶质瘤组织中的表达及其与p53/p14ARF信号通路失活的关系。方法实时定量PCR及Western Blot法检测8例正常脑组织及52例不同病理分级的脑胶质瘤组织中Wip1基因的表达,直接测序法检测p53基因的突变情况,逆转录PCR及甲基化PCR检测p14ARF的表达情况,统计学分析其相关性。结果52例胶质瘤样本中p53/p14ARF信号通路失活30例(57.7%)。22例无p53或p14ARF改变的样本中,11例伴有Wip1 mRNA过表达；而p53/p14ARF信号通路失活30例中,仅1例(3.3%)伴有Wip1 mRNA过表达。Wip1在脑胶质瘤中存在着选择性的过表达,过表达的Wip1主要与p53野生型相关而与p14ARF的甲基化状态及转录子表达无关。结论Wip1在脑胶质瘤中存在选择性的过表达；Wip1基因在脑胶质瘤中的过表达与p53/p14ARF信号通路的失活有关,Wip1基因可能抑制p53/p14ARF信号通路。目的构建人Wip1基因的RNA干扰(RNAi)慢病毒载体,测定病毒滴度,并对不同靶点进行筛选,挑选干扰效率最高的靶点。方法根据人Wip1基因序列,设计三对Wip1基因特异性RNAi靶序列,经退火制备双链DNA oligo,连接到pFU-GW-iRNA载体,转化后经PCR鉴定,阳性克隆测序证实,慢病毒包装转染293T细胞,获得病毒上清并测定其滴度。将病毒上清感染人胶质瘤细胞株U251和U-87MG,实时定量PCR及Western blot鉴定RNA干扰效率,筛选出基因沉默效率最高的慢病毒载体。结果PCR扩增和测序结果表明成功构建Wip1基因慢病毒干扰载体,慢病毒载体包装获得的病毒上清滴度在3-8×108TU/ml。可以有效地沉默U251细胞及U-87MG细胞中Wip1基因的表达,构建的RNA干扰慢病毒载体感染U251细胞后Wip1基因的mRNA表达量同对照组相比较分别为36.3%、32.9%、23.8%；在U-87MG中则分别为48.8%、36.7%和23.7%。Western blot结果显示Wip1蛋白表达量在感染细胞7d后显著下降。结论成功构建人Wip1基因的RNA干扰慢病毒载体并筛选出最佳靶点,为进一步研究Wip1基因在胶质瘤细胞中的作用提供可靠的实验平台。目的研究Wip1基因在人脑胶质瘤细胞恶性增殖中的作用及探讨其可能的作用机制。方法Wip1基因RNA干扰慢病毒载体感染胶质瘤细胞U251及U-87MG。CCK-8法(cell counting kit-8)及克隆形成实验检测细胞的增殖能力；流式细胞术Annexin V-APC/PI双标法及Hoechst 33258染色检测细胞的凋亡情况；流式细胞术PI单染法检测细胞周期分布；Transwell实验检测细胞的侵袭性；实时定量PCR法检测Wip1基因沉默后差异性基因的mRNA表达；Western blot检测Wip1可能作用途径的基因的蛋白表达。SPSS 13.0软件分析统计学差异。结果胶质瘤中Wip1基因的表达被有效沉默。Wip1基因沉默的胶质瘤细胞增殖变慢,在U-87MG细胞中更加明显；转染慢病毒载体的U-87MG和U251细胞在4d时凋亡无明显变化,但7d和10d时出现明显的凋亡,且凋亡率随着时间的延长而逐渐增加,U-87MG细胞的凋亡率为52.2%和67.1%,U251则分别为26.1%和38.2%。U-87MG在慢病毒感染后10d与U251细胞相比,凋亡率明显要高(P<0.05)；Hoechst 33258染色显示Wip1基因沉默后的胶质瘤细胞中可以观察到典型的细胞凋亡的形态学改变如核浓缩、核碎裂；细胞周期分析显示Wip1基因沉默对U251细胞周期无明显影响,而U-87MG细胞则在10d时出现明显的S期增多和Gl、G2期细胞的减少；U-87MG细胞在Wip1基因沉默后出现明显的体外侵袭力下降。Wip1基因沉默后,U251细胞PIK3R1基因出现上调,伴有CDKN2A和p14ARF基因的下调,而在U-87MG细胞中他们的表达均上调；Western blot结果显示两种胶质瘤细胞中的p38MAPK的表达均上调,而Akt的表达均下调,但是p-Akt(Ser473)却只在U-87MG细胞中存在上调。在U-87MG细胞中p53及p-p53 (Ser15)表达均上调,但在U251细胞中无明显变化。结论Wip1对胶质瘤细胞的增殖、凋亡和侵袭具有重要的作用,并且这种作用主要与其调节p53的功能有关。更多还原

【Abstract】 Objective Patients with gliomas often have mutations in the p53 gene. Wild-type p53-induced phosphatase 1 (Wip1 or PPM1D) encodes a negative regulator of p53. Wip1 over-expression inhibits p53 function and reduces selection for p53 mutations during cancer progression. Wip1 may have oncogenic function. To clarify the correlation of Wip1 with p53/p14ARF pathway disruption in glioma, the expression of Wip1 and p53/p14ARF pathway alterations have been investigated.Methods Tumor samples of 52 patients with primary glioams and 8 samples of normal brain tissues were examined for p53 mutations, p14ARF expression, and Wip1 expression. Direct sequencing of region from exons 5 to 8 of the p53 gene was performed on the genomic DNA in each tumor. The DNA methylation states of the CpG islands of the p14ARF gene were determined by methylation-specific PCR. All tumor specimens were analyzed for expression of Wip1 by real-time quantitative RCR, western blot and immunohistochemical staining.Results Disruption of the p53/p14ARF pathway was detected in 57.7%(30 in 52) of samples from patients with gliomas. Among the 22 cases without p53 and p14ARF alterations,11 (50%) had Wip1 mRNA over-expression. In tumors with p53 or p14ARF disruptions, Wip1 mRNA was over-expressed in only 1 case out of 30 (3.3%). Higher levels of Wip1 were associated with wild-type p53 but not with lower levels of expression of p14ARF or aberrant promoter hypermethylation of the p14ARF gene.Conclusion Wip1 is selectively over-expressed in tumors without alterations in p53 or p14ARF.Wip1 may inhibit the P53/p14ARF pathway.Objective To construct the lentiviral expression vector for RNA interference (RNAi) of Wip1 gene in glioma cells and select the optimal target sequence of Wip1 gene that is the most effective for RNAi.Methods Three double-stranded oligo DNAs were designed and synthesized according to the sequence of Wip1 gene and cloned into the pFU-GW-iRNA vector. After verification of the positive clones by PCR and sequence analysis, the verified plasmids were transfected into 293 T cells, the lenti virus was produced and the titer of virus was determined. The lentivirus was used to infect the glioma cell lines U251 and U-87MG. Real-time quantitative PCR and Western blot were performed to determine Wip1 expression levels in the virus infected glioma cells and the optimal interfering target was selected.Results Three recombinant lentiviral vector expressing shRNAs against Wip1 gene were obtained and confirmed by DNA sequencing. The titer of virus was 3-8×10 8TU/ml. Wip1 mRNA and protein expressions in U251 and U-87MG cells after infected with lentiviral vector were decreased remarkably. The mRNA expression in U251 cells was 36.3%,32.9% and 23.8% respectively, compared with the control group. And in U-87MG was 48.8%,36.7% and 23.7%. Western blot showed that Wip1 protein expression decreased 7 d after the infection significantly.Conclusion The lentiviral shRNA expression vector targeting human Wip1 gene capable of stable Wip1 gene silencing in glioma cells has been successfully constructed, which provides a basis for further study of mechanisms that Wip1 gene acts for malignant proliferation of glioma cells.Objective To detect the roles that Wip1 plays in the malignant growth of glioma cells and explore the possible mechanisms.Methods Glioma cells U251 and U-87MG were infected with Wip1 RNAi lentiviral vector. The proliferation activities of cells with Wip1 silencing were detected by cell counting kit-8 and clone formation experiments. Cell apoptosis was determined by flow cytometry AnnexinⅤ-APC/PI double labeling method and Hoechst 33258 staining. Cell cycle distributions were analyzed by flow cytometry PI staining. Cell invasiveness in glioma cells was determined by Transwell invasion assay. Real-time quantitative PCR was used to identify the mRNA expressions of differentially expressed genes after Wip1 silencing. Western blot was performed to detect the expressions of passageway proteins that Wipl may effect in. SPSS 13.0 software was used to analyze the significant difference.Results The expressions of Wip1gene in glioma cells were silenced effectively. Cells with Wip1 silencing, especially U-87MG cells, had reduced proliferation ability compared with mock cells. The number of apoptotic cells determined at 4 d after infection with Wip1 RNAi-R3 and NC lentiviral were not of the difference between them. At 7 d and 10 d, both U-87MG and U251 cells after Wip1 silencing showed significantly increased apoptotic cells compared with the mock cells. And the amount of apoptotic cells increased as the time periods extend. The apoptotic cells accounted for 52.2% and 67.1% of all the U-87MG cells with Wip1 silencing, while accounting for 26.1% and 38.2% in U251 cells. And U-87MG cells were more apoptotic than U251 cells 10 days after the RNAi-R3 lentiviral infection (P<0.05). Both U-87MG and U251 cells after Wip1 silencing had undergone extensive DNA strand breakage and nuclei-shrunk, characteristic changes of apoptosis. No significant changes of cell cycle distributions analyzed by flow cytometry were detected in U251 cells after Wip1 silencing. But increased S phase and decreased G1 and G2 phases were detected in U-87MG 10 d after the infection. U-87MG cells showed decreased invasiveness after Wip1 silencing. After Wip1 silencing, PIK3R1 gene mRNA expression was upregulated, while CDKN2A and p14ARF were downregulated in U251 cells. But in U-87MG cells, they all showed increased tendencies. Western blot showed that protein expression of p38MAPK in both U251 and U-87MG cells was increased, but expression of Akt was downregulated. Expression of p-Akt(Ser473)was only increased in U-87MG cells. The expressions of p53 and p-p53 (Ser 15) were both upregulated in U-87MG cell but remained unchanged in U251 cells.Conclusion Wip1 plays a important role in proliferation, apoptosis and invasiveness in glioma cells, and the effects are of much relevance with the fact that Wip1 could inhibit the function of p53.更多还原